Anti-apoc3 antibodies and methods of use thereof

ABSTRACT

The instant disclosure provides antibodies that specifically bind to ApoC3 (e.g., human ApoC3) and antagonize ApoC3 function. Also provided are pharmaceutical compositions comprising these antibodies, nucleic acids encoding these antibodies, expression vectors and host cells for making these antibodies, and methods of treating a subject using these antibodies.

RELATED APPLICATIONS

This application is a continuation of International Patent ApplicationNo. PCT/IB2018/052780, filed Apr. 20, 2018, which claims the benefit ofU.S. Provisional Patent Application Ser. No. 62/488,425, filed Apr. 21,2017, which are incorporated by reference herein in their entirety.

FIELD

The instant disclosure relates to antibodies that specifically bind toApoC3 (e.g., human ApoC3) and methods of using the same.

BACKGROUND

Elevated blood triglyceride levels (hypertriglyceridemia) are a causalfactor for atherosclerosis, and increase the risk of cardiovascularevents, such as cardiovascular death, angina, myocardial infarction, andstroke.

ApoC3 is a protein that circulates at very high concentrations (greaterthan 10 μM) in the blood, mostly bound to triglyceride rich lipoprotein(TRL), TRL remnants, and high density lipoprotein. ApoC3 appears to bean important regulator of blood triglyceride levels. For example, ApoC3levels in humans have been shown to positively correlate with bloodtriglyceride levels, with elevated ApoC3 levels being associated withhypertriglyceridemia. In addition, ApoC3 has been shown to inhibit theactivity of lipoprotein lipase (an enzyme that hydrolyses triglyceridesin TRL) and also to inhibit hepatic uptake of TRL remnants, both ofwhich cause elevation of blood triglyceride levels.

Several therapies have been approved for the treatmenthypertriglyceridemia, such as fibrates, niacin, and omega-3 fatty acids.However these therapies are only modestly effective at lowering plasmatriglycerides. Accordingly, there is a need in the art for improvedtherapies for lowering plasma triglycerides.

SUMMARY

The instant disclosure provides antibodies that specifically bind toApoC3 (e.g., human ApoC3) and inhibit ApoC3 function. Also provided arepharmaceutical compositions comprising these antibodies, nucleic acidsencoding these antibodies, expression vectors and host cells for makingthese antibodies, and methods of treating a subject using theseantibodies.

In certain embodiments, the anti-ApoC3 antibodies disclosed herein canattenuate the ability of ApoC3 to inhibit TRL uptake by hepatocytes andcan cause a rapid and sustained decrease in the serum levels of ApoC3and ApoB when administered to a subject. Accordingly, the disclosedanti-ApoC3 antibodies are useful for the treatment and prevention ofhypertriglyceridemia and associated diseases (e.g., cardiovasculardisease and pancreatitis).

Accordingly, in one aspect, the instant disclosure provides an isolatedantibody that specifically binds to ApoC3 with a first dissociationconstant (K_(D)) at pH 7.4 and with a second K_(D) at pH 5.5, whereinthe ratio between the second K_(D) and the first K_(D) is greater thanor at least about 5, 10, 20, or 50. In certain embodiments, the firstK_(D) is less than 10, 5, 2, 1, 0.5, 0.2, or 0.1 nM. In certainembodiments, the half-life of the antibody in a mouse expressing ApoC3is greater than or at least about 3, 7, 14, 21, or 28 days.

In certain embodiments, the antibody attenuates the ability of ApoC3 toinhibit hepatocyte uptake of very low density lipoprotein (VLDL). Incertain embodiments, the antibody is capable of increasing the rate ofclearance of ApoC3 from the blood in a subject. In certain embodiments,the antibody is capable of increasing the rate of clearance of ApoB fromthe blood in a subject. In certain embodiments, the antibody is capableof reducing the level of ApoC3 in the blood in a subject. In certainembodiments, the antibody is capable of reducing the level of ApoC3 inthe blood in a subject by at least 40% for at least 2 weeks. In certainembodiments, the antibody is capable of reducing the level of ApoB inthe blood in a subject. In certain embodiments, the antibody is capableof reducing the level of ApoB in the blood in a subject by at least 20%for at least 2 weeks. In certain embodiments, the antibody is capable ofinhibiting post-prandial lipemia in a subject. In certain embodiments,the antibody is capable of binding to lipid-bound ApoC3.

In certain embodiments, the antibody binds to an epitope within theamino acid sequence set forth in SEQ ID NO: 2. In certain embodiments,the epitope comprises at least one of the amino acids at position 2, 5,6, 8, or 10 of SEQ ID NO: 2. In certain embodiments, the epitopecomprises the amino acids at positions 5 and 6 of SEQ ID NO: 2. Incertain embodiments, the epitope comprises the amino acids at positions2, 5, 6, and 8 of SEQ ID NO: 2. In certain embodiments, the epitopecomprises the amino acids at position 10 of SEQ ID NO: 2. In certainembodiments, the epitope comprises the amino acids at positions 6, 8,and 10 of SEQ ID NO: 2. In certain embodiments, the epitope comprisesthe amino acids at positions 6 and 8 of SEQ ID NO: 2.

In certain embodiments, the antibody comprises a heavy chain variableregion having complementarity determining regions CDRH1, CDRH2 andCDRH3, and a light chain variable region having complementaritydetermining regions CDRL1, CDRL2 and CDRL3, and wherein:

(a) CDRH1 comprises the amino acid sequence of TYSMR (SEQ ID NO: 3);(b) CDRH2 comprises the amino acid sequence of SIX₁TDGGGTAYRDSVKG,wherein X₁ is S or H (SEQ ID NO: 4);(c) CDRH3 comprises the amino acid sequence of X₂GYSD, wherein X₂ is Aor H (SEQ ID NO: 5);(d) CDRL1 comprises the amino acid sequence of KTSQGLVHSDGKTYFY (SEQ IDNO: 6);(e) CDRL2 comprises the amino acid sequence of QVSNRAS (SEQ ID NO: 7);and(f) CDRL3 comprises the amino acid sequence of AX₃GTYYPHT, wherein X₃ isQ or H (SEQ ID NO: 8), and wherein at least one of X₁, X₂, and X₃ is H.

In certain embodiments, the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3comprise the amino acid sequences set forth in SEQ ID NOs: 3, 11, 10, 6,7, and 13; 3, 9, 12, 6, 7, and 13; 3, 9, 10, 6, 7, and 14; 3, 11, 10, 6,7, and 14; 3, 9, 12, 6, 7, and 14; 3, 11, 12, 6, 7, and 13; or 3, 11,12, 6, 7, and 13, respectively. In certain embodiments, the heavy chainvariable region comprises an amino acid sequences selected from thegroup consisting of SEQ ID NOs: 16-18. In certain embodiments, the lightchain variable region comprises the amino acid sequence set forth in SEQID NO: 20. In certain embodiments, the heavy chain variable region andthe light chain variable region, respectively, comprise the amino acidsequences set forth in SEQ ID NOs: 16 and 19, 17 and 19, 18 and 19, 15and 20, 16 and 20, 17 and 20, or 18 and 20, respectively.

In another aspect, the instant disclosure provides an isolated antibodythat specifically binds to ApoC3, the antibody comprising a heavy chainvariable region having complementarity determining regions CDRH1, CDRH2and CDRH3, and a light chain variable region having complementaritydetermining regions CDRL1, CDRL2 and CDRL3, wherein:

(a) CDRH1 comprises the amino acid sequence of TYSMR (SEQ ID NO: 3);(b) CDRH2 comprises the amino acid sequence of SIX₁TDGGGTAYRDSVKG,wherein X₁ is S or H (SEQ ID NO: 4);(c) CDRH3 comprises the amino acid sequence of X₂GYSD, wherein X₂ is Aor H (SEQ ID NO: 5);(d) CDRL1 comprises the amino acid sequence of KTSQGLVHSDGKTYFY (SEQ IDNO: 6);(e) CDRL2 comprises the amino acid sequence of QVSNRAS (SEQ ID NO: 7);and(f) CDRL3 comprises the amino acid sequence of AX₃GTYYPHT, wherein X₃ isQ or H (SEQ ID NO: 8), and wherein at least one of X₁, X₂, and X₃ is H.

In certain embodiments, the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3comprise the amino acid sequences set forth in SEQ ID NOs: 3, 11, 10, 6,7, and 13; 3, 9, 12, 6, 7, and 13; 3, 9, 10, 6, 7, and 14; 3, 11, 10, 6,7, and 14; 3, 9, 12, 6, 7, and 14; 3, 11, 12, 6, 7, and 13; or 3, 11,12, 6, 7, and 13, respectively.

In another aspect, the instant disclosure provides an isolated antibodythat specifically binds to ApoC3, the antibody comprising a heavy chainvariable region comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 16-18.

In another aspect, the instant disclosure provides an isolated antibodythat specifically binds to ApoC3, the antibody comprising a light chainvariable region comprising the amino acid sequence set forth in SEQ IDNO: 20.

In another aspect, the instant disclosure provides an isolated antibodythat specifically binds to ApoC3, the antibody comprising a heavy chainvariable region and a light chain variable region, wherein the heavychain variable region and the light chain variable region, respectively,comprise the amino acid sequences set forth in SEQ ID NOs: 16 and 19, 17and 19, 18 and 19, 15 and 20, 16 and 20, 17 and 20, or 18 and 20,respectively.

In certain embodiments of any one of the foregoing aspects, the antibodyfurther comprises a constant region (e.g., a human or humanized constantregion). In certain embodiments, the constant region is a variant of awild type human immunoglobulin heavy chain constant region, and whereinthe variant human immunoglobulin heavy chain constant region has anincreased affinity for human neonatal Fc receptor (FcRn) at pH 6relative to the affinity of the wild type human immunoglobulin heavychain constant region for human FcRn at pH 6.

In certain embodiments, the constant region is a heavy chain constantregion of a human IgG. In certain embodiments, the constant region is aheavy chain constant region of a human IgG₁, IgG₂, or IgG₄. In certainembodiments, the constant region comprises the amino acids K, F, and Yat EU positions 433, 434, and 436, respectively. In certain embodiments,the constant region comprises the amino acids Y, T, and E at EUpositions 252, 254, and 256, respectively. In certain embodiments, theconstant region comprises the amino acids L and S at EU positions 428and 434, respectively. In certain embodiments, the constant regioncomprises an amino acid sequence selected from the group consisting ofSEQ ID NOs: 22-24, 37-39, and 42-47.

In certain embodiments of any one of the aspects disclosed herein, ApoC3is human ApoC3.

In another aspect, the instant disclosure provides a pharmaceuticalcomposition comprising an antibody as disclosed herein and apharmaceutically acceptable carrier.

In another aspect, the instant disclosure provides a polynucleotideencoding the heavy chain variable region or the light chain variableregion of an antibody as disclosed herein.

In another aspect, the instant disclosure provides an expression vectorcomprising the polynucleotide as disclosed herein. In another aspect,the instant disclosure provides a host cell comprising the expressionvector as disclosed herein.

In another aspect, the instant disclosure provides a method forproducing an antibody that binds to ApoC3, the method comprisingculturing the host cell as disclosed herein under conditions that allowexpression of the antibody.

In another aspect, the instant disclosure provides a method forinhibiting the activity of ApoC3 in a subject, the method comprisingadministering to the subject an effective amount of an antibody orpharmaceutical composition as disclosed herein. In another aspect, theinstant disclosure provides a method for reducing triglyceride levels inthe blood of a subject, the method comprising administering to thesubject an effective amount of an antibody or pharmaceutical compositionas disclosed herein. In another aspect, the instant disclosure providesa method for inhibiting post-prandial lipemia in a subject, the methodcomprising administering to the subject an effective amount of anantibody or pharmaceutical composition as disclosed herein. In anotheraspect, the instant disclosure provides a method for treatinghypertriglyceridemia in a subject, the method comprising administeringto the subject an effective amount of an antibody or pharmaceuticalcomposition as disclosed herein. In another aspect, the instantdisclosure provides a method for treating chylomicronemia in a subject,the method comprising administering to the subject an effective amountof an antibody or pharmaceutical composition as disclosed herein.

In another aspect, the instant disclosure provides a method for reducingthe risk of cardiovascular disease in a subject withhypertriglyceridemia, the method comprising administering to the subjectan effective amount of an antibody or pharmaceutical composition asdisclosed herein. In certain embodiments, the cardiovascular disease ismyocardial infarction. In certain embodiments, the cardiovasculardisease is angina. In certain embodiments, the cardiovascular disease isstroke. In certain embodiments, the cardiovascular disease isatherosclerosis.

In certain embodiments of the foregoing aspects relating to treatmentmethods, the antibody reduces the levels of chylomicron or chylomicronremnants in the blood of the subject. In certain embodiments, thesubject is receiving an additional lipid lowering agent. In certainembodiments, the additional lipid lowering agent is an HMG-CoA reductaseinhibitor. In certain embodiments, the HMG-CoA reductase inhibitor isatorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin,rosuvastatin or simvastatin. In certain embodiments, the additionallipid lowering agent is a PCSK9 inhibitor. In certain embodiments, thePCSK9 inhibitor is alirocumab, evolocumab, or bococizumab. In certainembodiments, the additional lipid lowering agent is ezetimibe. Incertain embodiments, the additional lipid lowering agent is acombination of ezetimibe and an HMG-CoA reductase inhibitor. In certainembodiments, the additional lipid lowering agent is a combination ofezetimibe, an HMG-CoA reductase inhibitor, and a PCSK9 inhibitor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B, and 1C are a series of graphs showing that the 5E5WT (FIG.1A), 5E5VH5_VL8 (FIG. 1B), and 5E5VHWT_VL8 (“VL8”), 5E5VH12_VLWT(“VH12”), 5E5VH5_VLWT (“VH5”), and 5E5VH5_VL8 (“VH5_VL8”) (FIG. 1C)antibodies attenuated the ability of ApoC3 to inhibit very low densitylipoprotein (VLDL) uptake by HepG2 cells. HepG2 cells were incubatedwith DiI VLDL and purified ApoC3 either alone or in the presence of ananti-ApoC3 antibody as indicated. DiI VLDL ingested by HepG2 cells weremeasured by fluorescence spectroscopy of the DiI dye. HepG2 cellsincubated with DiI VLDL alone (“VLDL”) served as a positive control, andHepG2 cells incubated with DiI VLDL and purified ApoC3 in the absence ofan anti-ApoC3 antibody (“ApoC3”) served as a negative control.

FIGS. 2A-2C are graphs showing pharmacokinetics and pharmacodynamics oftwo anti-ApoC3 antibodies, 5E5 and 5E5VH5_VL8, and an anti-hen egglysosome human IgG₁ antibody (HyHe15), in an AAV8-human ApoC3 mousemodel. The test antibodies were administered intravenously to miceexpressing transgenic human ApoC3. The serum levels of human IgG₁ (FIG.2A), human ApoC3 (FIG. 2B), and mouse ApoB (FIG. 2C) at various timespost-injection were measured, and the absolute or relative levelscompared to time 0 were plotted.

FIGS. 3A-3D are graphs showing reduction of fasting triglyceride levelsand circulating post-prandial triglyceride levels by 5E5VH5_VL8 in anAAV8-human ApoC3 mouse model (n=6 per treatment group). The plasmatriglyceride levels in mice treated with 5E5VH5_VL8 or the HyHe15antibody before and after fasting overnight are shown in FIG. 3A. Theplasma triglyceride levels in these mice after an olive oil challengeare shown in FIG. 3B, and the calculated area under the curve values areplotted in FIG. 3C. The ApoC3 levels throughout the course of antibodytreatment, fasting, and olive oil challenge are plotted in FIG. 3D.

DETAILED DESCRIPTION

The instant disclosure provides antibodies that specifically bind toApoC3 (e.g., human ApoC3) and inhibit ApoC3 function. Also provided arepharmaceutical compositions comprising these antibodies, nucleic acidsencoding these antibodies, expression vectors and host cells for makingthese antibodies, and methods of treating a subject using theseantibodies. In certain embodiments, the anti-ApoC3 antibodies disclosedherein can attenuate the ability of ApoC3 to inhibit TRL uptake byhepatocytes and can cause a rapid and sustained decrease in the serumlevels of ApoC3 and ApoB when administered to a subject. Accordingly,the disclosed anti-ApoC3 antibodies are useful for the treatment andprevention of hypertriglyceridemia and associated diseases (e.g.,cardiovascular disease and pancreatitis).

1. Definitions

As used herein, the term “ApoC3” refers to Apolipoprotein C3 protein. Incertain embodiments, the ApoC3 is human ApoC3. An exemplary human ApoC3amino acid sequence is set forth in RefSeq accession number NP_000031.1.The mature amino acid sequence of NP_000031.1 is as follows:

(SEQ ID NO: 1) SEAEDASLLSFMQGYMKHATKTAKDALSSVQESQVAQQARGWVTDGFSSLKDYWSTVKDKFSEFWDLDPEVRPTSAVAA.

As used herein, the terms “antibody” and “antibodies” include fulllength antibodies, antigen-binding fragments of full length antibodies,and molecules comprising antibody CDRs, VH regions or VL regions.Examples of antibodies include monoclonal antibodies, recombinantlyproduced antibodies, monospecific antibodies, multispecific antibodies(including bispecific antibodies), human antibodies, humanizedantibodies, chimeric antibodies, immunoglobulins, synthetic antibodies,tetrameric antibodies comprising two heavy chain and two light chainmolecules, an antibody light chain monomer, an antibody heavy chainmonomer, an antibody light chain dimer, an antibody heavy chain dimer,an antibody light chain-antibody heavy chain pair, intrabodies,heteroconjugate antibodies, single domain antibodies, monovalentantibodies, single chain antibodies or single-chain Fvs (scFv),scFv-Fcs, camelid antibodies (e.g., llama antibodies), camelizedantibodies, affybodies, Fab fragments, F(ab′)₂ fragments,disulfide-linked Fvs (sdFv), anti-idiotypic (anti-Id) antibodies(including, e.g., anti-anti-Id antibodies), and antigen-bindingfragments of any of the above. In certain embodiments, antibodiesdisclosed herein refer to polyclonal antibody populations. Antibodiescan be of any type (e.g., IgG, IgE, IgM, IgD, IgA or IgY), any class(e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁ or IgA₂), or any subclass (e.g.,IgG₂a or IgG₂b) of immunoglobulin molecule. In certain embodiments,antibodies disclosed herein are IgG antibodies, or a class (e.g., humanIgG₁ or IgG₄) or subclass thereof. In a specific embodiment, theantibody is a humanized monoclonal antibody.

As used herein, the term “isolated antibody” refers to an antibody thathas been identified and separated and/or recovered from at least onecomponent of its natural environment. The term “isolated antibody”includes an antibody in situ within a recombinant host cell.

As used herein, the term “CDR” or “complementarity determining region”means the noncontiguous antigen combining sites found within thevariable region of both heavy and light chain polypeptides. Theseparticular regions have been described by Kabat et al., J. Biol. Chem.252, 6609-6616 (1977) and Kabat et al., Sequences of protein ofimmunological interest. (1991), by Chothia et al., J. Mol. Biol.196:901-917 (1987), and by MacCallum et al., J. Mol. Biol. 262:732-745(1996), all of which are incorporated by reference in their entireties,where the definitions include overlapping or subsets of amino acidresidues when compared against each other. In certain embodiments, theterm “CDR” is a CDR as defined by Kabat et al., J. Biol. Chem. 252,6609-6616 (1977) and Kabat et al., Sequences of protein of immunologicalinterest. (1991). CDRH1, CDRH2 and CDRH3 denote the heavy chain CDRs,and CDRL1, CDRL2 and CDRL3 denote the light chain CDRs.

As used herein, the term “framework (FR) amino acid residues” refers tothose amino acids in the framework region of an immunoglobulin chain.The term “framework region” or “FR region” as used herein, includes theamino acid residues that are part of the variable region, but are notpart of the CDRs (e.g., using the Kabat definition of CDRs).

As used herein, the terms “variable region” and “variable domain” areused interchangeably and are common in the art. The variable regiontypically refers to a portion of an antibody, generally, a portion of alight or heavy chain, typically about the amino-terminal 110 to 120amino acids or 110 to 125 amino acids in the mature heavy chain andabout 90 to 115 amino acids in the mature light chain, which differextensively in sequence among antibodies and are used in the binding andspecificity of a particular antibody for its particular antigen. Thevariability in sequence is concentrated in those regions calledcomplementarity determining regions (CDRs) while the more highlyconserved regions in the variable domain are called framework regions(FR). Without wishing to be bound by any particular mechanism or theory,it is believed that the CDRs of the light and heavy chains are primarilyresponsible for the interaction and specificity of the antibody withantigen. In certain embodiments, the variable region is a human variableregion. In certain embodiments, the variable region comprises rodent ormurine CDRs and human framework regions (FRs). In particularembodiments, the variable region is a primate (e.g., non-human primate)variable region. In certain embodiments, the variable region comprisesrodent or murine CDRs and primate (e.g., non-human primate) frameworkregions (FRs).

The terms “VL” and “VL domain” are used interchangeably to refer to thelight chain variable region of an antibody.

The terms “VH” and “VH domain” are used interchangeably to refer to theheavy chain variable region of an antibody.

As used herein, the terms “constant region” and “constant domain” areinterchangeable and are common in the art. The constant region is anantibody portion, e.g., a carboxyl terminal portion of a light or heavychain which is not directly involved in binding of an antibody toantigen but which can exhibit various effector functions, such asinteraction with the Fc receptor. The constant region of animmunoglobulin molecule generally has a more conserved amino acidsequence relative to an immunoglobulin variable domain.

As used herein, the term “heavy chain” when used in reference to anantibody can refer to any distinct type, e.g., alpha (α), delta (δ),epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence ofthe constant domain, which give rise to IgA, IgD, IgE, IgG, and IgMclasses of antibodies, respectively, including subclasses of IgG, e.g.,IgG₁, IgG₂, IgG₃, and IgG₄.

As used herein, the term “light chain” when used in reference to anantibody can refer to any distinct type, e.g., kappa (κ) or lambda (λ)based on the amino acid sequence of the constant domains. Light chainamino acid sequences are well known in the art. In specific embodiments,the light chain is a human light chain.

As used herein, the term “EU position” refers to the amino acid positionaccording to the EU numbering convention for the constant regions of anantibody, as described in Edelman, G. M. et al., Proc. Natl. Acad. USA,63, 78-85 (1969) and Kabat et al., in “Sequences of Proteins ofImmunological Interest”, U.S. Dept. Health and Human Services, 5thedition, 1991, each of which is herein incorporated by reference in itsentirety.

As used herein, the term “specifically binds to” refers to the abilityof an antibody to bind to an antigen with an dissociation constant(K_(D)) of less than about 1×10⁻⁶ M, 1×10⁻⁷ M, 1×10⁻⁸ M, 1×10⁻⁹ M,1×10⁻¹⁰ M, 1×10⁻¹¹ M, 1×10⁻¹² M, or less, or bind to an antigen with anaffinity that is at least two-fold greater than its affinity for anonspecific antigen.

As used herein, an “epitope” refers to a localized region of an antigento which an antibody can specifically bind. An epitope can be, forexample, contiguous amino acids of a polypeptide (a linear or contiguousepitope) or an epitope can, for example, be formed from two or morenon-contiguous regions of a polypeptide or polypeptides (aconformational, non-linear, discontinuous, or non-contiguous epitope).In certain embodiments, the epitope to which an antibody binds can bedetermined by, e.g., NMR spectroscopy, X-ray diffraction crystallographystudies, ELISA assays, hydrogen/deuterium exchange coupled with massspectrometry (e.g., liquid chromatography electrospray massspectrometry), peptide scanning assays, or mutagenesis mapping (e.g.,site-directed mutagenesis mapping).

As used herein, the term “treat,” “treating,” and “treatment” refer totherapeutic or preventative measures disclosed herein. The methods of“treatment” employ administration of an anti-ApoC3 antibody to a subjecthaving a disease or disorder, or predisposed to having such a disease ordisorder, in order to prevent, cure, delay, reduce the severity of,reduce the risk of developing, or ameliorate one or more symptoms of thedisease or disorder or recurring disease or disorder, or in order toprolong the survival of a subject beyond that expected in the absence ofsuch treatment.

As used herein, the term “effective amount” in the context of theadministration of a therapy to a subject refers to the amount of atherapy that achieves a desired prophylactic or therapeutic effect.

As used herein, the term “subject” includes any human or non-humananimal.

As used herein, the term “or” means and/or.

As used herein, the terms “about” and “approximately,” when used tomodify a numeric value or numeric range, indicate that deviations of 5%to 10% above and 5% to 10% below the value or range remain within theintended meaning of the recited value or range.

2. Anti-ApoC3 Antibodies

The instant disclosure provides isolated antibodies that specificallybind to ApoC3 (e.g., human ApoC3) and inhibit ApoC3 function.

In certain embodiments, the isolated antibodies bind to ApoC3 protein ofa mammal. In certain embodiments, the isolated antibodies bind to humanApoC3. In certain embodiments, the isolated antibodies bind to Macacafascicularis (cynomolgus monkey) ApoC3.

In certain embodiments, the isolated antibodies bind to ApoC3 (e.g.,human ApoC3) with a higher affinity at physiological pH (e.g., pH 7.4)than under at acidic pH (e.g., pH 5.5 to pH 6). Methods for generatingsuch pH-dependent antibodies are well known in the art. For example, inone exemplary method, one or more amino residues in the heavy and/orlight chain CDRs of an anti-ApoC3 antibody are substituted with ahistidine residue, as described in: Igawa et al., Nat Biotechnol. (2010)28(11):1203-1207; Chaparro-Riggers et al., J Biol Chem. (2012)287(14):11090-11097; U.S. Pat. No. 9,096,651, and U.S. patentpublication number US20110111406A1, each of which is hereby incorporatedby reference herein in its entirety. However, although such methods arewell known in the art, the skilled worker will appreciate that, for anygiven antibody, the precise CDR amino acids that can be mutated tohistidine to achieve pH-dependent binding to antigen without disruptingthe antibody's affinity for antigen can only be determined empirically(see e.g., Edgcomb and Murphy, Proteins (2002) 49:1-6, which is herebyincorporated by reference herein in its entirety).

A skilled person in the art would appreciate that the affinity of anantibody to an antigen can be indicated by the dissociation constant(K_(D)), wherein a smaller K_(D) indicates a higher affinity.Accordingly, in certain embodiments, the anti-ApoC3 antibodies bind toApoC3 (e.g., human ApoC3) with a first K_(D) at pH 7.4 and with a secondK_(D) at pH 5.5, wherein the ratio between the second K_(D) and thefirst K_(D) is greater than or at least 1 (e.g., greater than or atleast 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4,4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50,60, 70, 80, 90, or 100).

In certain embodiments, the first K_(D) is less than 100 nM (e.g., lessthan 50, 20, 10, 5, 2, 1, 0.5, 0.2, or 0.1 nM). In certain embodiments,the second K_(D) is greater than 1 nM (e.g., greater than 2, 5, 10, 20,or 50 nM, or greater than 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50, or 100μM). In certain embodiments, the first K_(D) is less than 100 nM (e.g.,less than 50, 20, 10, 5, 2, 1, 0.5, 0.2, or 0.1 nM), and the half-lifeof the antibody in an animal (e.g., a human or a mouse) expressing ApoC3(e.g., human ApoC3) is greater than or at least about 1 day (e.g.,greater than or at least about 2, 3, 4, 5, 6, or 7 days, or greater thanabout 1, 2, 3, 4, 6, or 8 weeks). In certain embodiments, the ApoC3 ishuman ApoC3, and the animal expressing the ApoC3 is a human. In certainembodiments, the ApoC3 is human ApoC3, and the animal expressing theApoC3 is a mouse expressing human ApoC3.

In certain embodiments, the isolated antibodies disclosed hereinattenuate the ability of ApoC3 to inhibit hepatocyte uptake of TRL(e.g., VLDL) or TRL remnants (in vivo or in vitro). In certainembodiments, the isolated antibodies disclosed herein attenuate theability of ApoC3 to inhibit hepatocyte uptake of TRL (e.g., VLDL) or TRLremnants by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, as assessed bymethods disclosed herein or by methods known to one of skill in the art.In certain embodiments, the isolated antibodies disclosed hereinattenuate the ability of ApoC3 to inhibit hepatocyte uptake of TRL(e.g., VLDL) or TRL remnants by at least about 1.1 fold, 1.2 fold, 1.3fold, 1.4 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold,4.5 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, or100 fold, as assessed by methods disclosed herein or by methods known toone of skill in the art.

In certain embodiments, the isolated antibodies disclosed herein arecapable of inhibiting post-prandial lipemia in a subject whenadministered to the subject prior to, during, or after a meal. Incertain embodiments, the anti-ApoC3 antibodies disclosed herein arecapable of inhibiting post-prandial lipemia in the subject by at least5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 98%, or 99%, as assessed by methods disclosedherein or by methods known to one of skill in the art. In certainembodiments, the anti-ApoC3 antibodies disclosed herein are capable ofinhibiting post-prandial lipemia in the subject by at least about 1.1fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold,3.5 fold, 4 fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80fold, 90 fold, or 100 fold, as assessed by methods disclosed herein orby methods known to one of skill in the art.

In certain embodiments, the isolated antibodies disclosed herein arecapable of reducing the levels of post-prandial chylomicron orchylomicron remnants in a subject when administered to the subject priorto, during, or after a meal. In certain embodiments, the anti-ApoC3antibodies disclosed herein are capable of reducing the levels ofpost-prandial chylomicron or chylomicron remnants in a subject by atleast 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, as assessed by methodsdisclosed herein or by methods known to one of skill in the art. Incertain embodiments, the anti-ApoC3 antibodies disclosed herein arecapable of reducing the levels of post-prandial chylomicron orchylomicron remnants in a subject by at least about 1.1 fold, 1.2 fold,1.3 fold, 1.4 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90fold, or 100 fold, as assessed by methods disclosed herein or by methodsknown to one of skill in the art.

In certain embodiments, the isolated antibodies disclosed herein arecapable of increasing the rates of clearance of ApoC3 and/or ApoB (e.g.,ApoB48 and/or ApoB100) from the blood in a subject. In certainembodiments, the anti-ApoC3 antibodies are capable of increasing therates of clearance of ApoC3 and/or ApoB (e.g., ApoB48 and/or ApoB100)from the blood in a subject by at least 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or99%, as assessed by methods disclosed herein or by methods known to oneof skill in the art. In certain embodiments, the anti-ApoC3 antibodiesdisclosed herein are capable of increasing the rates of clearance ofApoC3 and/or ApoB (e.g., ApoB48 and/or ApoB100) from the blood in asubject by at least about 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 6fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, or 100 fold, asassessed by methods disclosed herein or by methods known to one of skillin the art. Methods for assessing the clearance of ApoC3 and/or ApoB(e.g., ApoB48 and/or ApoB100) include without limitation the isotopetracer techniques, wherein the isotope can be either radioactive orstable.

In certain embodiments, the isolated antibodies disclosed herein arecapable of reducing the levels of ApoC3 and/or ApoB (e.g., ApoB48 and/orApoB100) in the blood in a subject. In certain embodiments, theanti-ApoC3 antibodies are capable of reducing the levels of ApoC3 and/orApoB (e.g., ApoB48 and/or ApoB100) in the blood in a subject by at least5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 98%, or 99%, as assessed by methods disclosedherein or by methods known to one of skill in the art. In certainembodiments, the anti-ApoC3 antibodies disclosed herein are capable ofreducing the levels of ApoC3 and/or ApoB (e.g., ApoB48 and/or ApoB100)in the blood in a subject by at least about 1.1 fold, 1.2 fold, 1.3fold, 1.4 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold,4.5 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, or100 fold, as assessed by methods disclosed herein or by methods known toone of skill in the art. In certain embodiments, the reduction in thelevels of ApoC3 and/or ApoB (e.g., ApoB48 and/or ApoB100) in the bloodin the subject is maintained for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 25, 30, 35, 40, 45, or 50 days, or at least 1, 2, 3, 4, 5, 6, 7,or 8 weeks.

In certain embodiments, the isolated antibodies disclosed herein arecapable of binding to lipid-bound ApoC3 (e.g., ApoC3 bound totriglyceride, TRL (e.g., VLDL) or TRL remnants). In certain embodiments,the isolated antibodies disclosed herein do not inhibit the binding ofApoC3 to a lipid or a lipoprotein. In certain embodiments, theantibodies disclosed herein do not compete for the binding of ApoC3 witha lipid or a lipoprotein. In certain embodiments, the lipid comprises afatty acid chain. In certain embodiments, the lipid comprises aphosphatidyl group. In certain embodiments, the lipid comprises aphosphatidylcholine (e.g., DMPC), a phosphatidylserine, aphosphatidylethanolamine, a phosphatidylinositol or aphosphatidylglycerol. In certain embodiments, the lipid is atriglyceride. In certain embodiments, the lipoprotein is a TRL (e.g.,VLDL) or a TRL remnant. In certain embodiments, the ability of ApoC3 tobind to lipids and lipoproteins (e.g., triglyceride, TRL (e.g., VLDL) orTRL remnants) in the presence of an anti-ApoC3 antibody disclosed hereinis at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or99% of the ability of ApoC3 to bind to the same lipids and lipoproteinsin the absence of an anti-ApoC3 antibody, as assessed by methodsdisclosed herein or by methods known to one of skill in the art.

In certain embodiments, the isolated antibodies disclosed hereinattenuate the ability of ApoC3 to inhibit hepatocyte uptake of TRL(e.g., VLDL) or TRL remnants. In certain embodiments, the uptake of TRL(e.g., VLDL) or TRL remnants by hepatocytes (e.g., HepG2 cells) in thepresence of an anti-ApoC3 antibody as disclosed herein is at least 1.1,1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, or 5folds higher than the uptake of TRL (e.g., VLDL) or TRL remnants byhepatocytes (e.g., HepG2 cells) in the absence of an anti-ApoC3antibody.

In certain embodiments, the isolated antibodies disclosed hereinattenuate the ability of ApoC3 to inhibit hepatocyte uptake of TRL(e.g., VLDL) or TRL remnants, and are capable of binding to lipid-boundApoC3 (e.g., ApoC3 bound to triglyceride, TRL (e.g., VLDL) or TRLremnants.

In certain embodiments, the isolated antibodies disclosed herein bind toan epitope of ApoC3 within the amino acid sequence FSEFWDLDPE (SEQ IDNO: 2). In certain embodiments, the epitope comprises at least one aminoacid within SEQ ID NO: 2, and optionally comprises one or more aminoacids from SEQ ID NO: 1 contiguous to SEQ ID NO: 2. In certainembodiments, the epitope comprises at least one of the amino acid atposition 2, 5, 6, 8, or 10 of SEQ ID NO: 2. In certain embodiments, theepitope comprises at least two of the amino acid at position 2, 5, 6, 8,or 10 of SEQ ID NO: 2. In certain embodiments, the epitope comprises atleast three of the amino acid at position 2, 5, 6, 8, or 10 of SEQ IDNO: 2. In certain embodiments, the epitope comprises at least four ofthe amino acid at position 2, 5, 6, 8, or 10 of SEQ ID NO: 2. In certainembodiments, the epitope comprises the amino acids at positions 5 and 6of SEQ ID NO: 2. In certain embodiments, the epitope comprises the aminoacids at positions 2, 5 and 6 of SEQ ID NO: 2. In certain embodiments,the epitope comprises the amino acids at positions 2, 5 and 8 of SEQ IDNO: 2. In certain embodiments, the epitope comprises the amino acids atpositions 2, 5, 6, and 8 of SEQ ID NO: 2. In certain embodiments, theepitope comprises the amino acid at position 10 of SEQ ID NO: 2. Incertain embodiments, the epitope comprises the amino acids at positions6 and 10 of SEQ ID NO: 2. In certain embodiments, the epitope comprisesthe amino acids at positions 8 and 10 of SEQ ID NO: 2. In certainembodiments, the epitope comprises the amino acids at positions 6 and 8of SEQ ID NO: 2. In certain embodiments, the epitope comprises the aminoacids at positions 6, 8 and 10 of SEQ ID NO: 2. In certain embodiments,the antibodies are capable of binding to lipid-bound ApoC3 (e.g., ApoC3bound to triglyceride, TRL (e.g., VLDL) or TRL remnants). In certainembodiments, the antibodies are not capable of attenuating the abilityof ApoC3 to inhibit lipoprotein lipase-mediated lipolysis of TRL (e.g.,VLDL). In certain embodiments, the antibodies also attenuate the abilityof ApoC3 to inhibit hepatocyte uptake of TRL (e.g., VLDL) or TRLremnants. In certain embodiments, the antibodies are also capable ofinhibiting post-prandial lipemia in a subject when administered to thesubject prior to, during, or after a meal. In certain embodiments, theantibodies disclosed herein are also capable of reducing the levels ofpost-prandial chylomicron or chylomicron remnants in a subject whenadministered to the subject prior to, during, or after a meal.

Any suitable assays can be used to measure the foregoing functionalactivities of the antibodies disclosed herein. Exemplary assays include,but are not limited to, the functional assays disclosed in the Examplesherein.

The amino acid sequences of exemplary anti-ApoC3 antibodies are setforth in Tables 1-7, herein.

TABLE 1 Heavy chain CDR amino acid sequences of exemplaryanti-ApoC3 antibodies. SEQ SEQ SEQ ID ID ID VH clone CDRH1 NO CDRH2 NOCDRH3 NO 5E5VHWT TYSMR 3 SISTDGGGTAYRDSVKG 9 AGYSD 10 5E5VH5 TYSMR 3SIHTDGGGTAYRDSVKG 11 AGYSD 10 5E5VH12 TYSMR 3 SISTDGGGTAYRDSVKG 9 HGYSD12 5E5VH5VH12 TYSMR 3 SIHTDGGGTAYRDSVKG 11 HGYSD 12

TABLE 2 Light chain CDR amino acid sequences ofexemplary anti-ApoC3 antibodies. SEQ SEQ SEQ VL ID ID ID clone CDRL1 NOCDRL2 NO CDRL3 NO 5E5VLWT KTSQGLVHSDGKTYFY 6 QVSNRAS 7 AQGTYYPHT 135E5VL8 KTSQGLVHSDGKTYFY 6 QVSNRAS 7 AHGTYYPHT 14

TABLE 3 VH amino acid sequences of exemplary anti-ApoC3 antibodies.SEQ ID VH clone Amino acid Sequence NO 5E5VHWTQLQLVESGGGLVQPGGSLRLSCAASGFTFGTYSMRWVRQVP 15RKALEWVSSISTDGGGTAYRDSVKGRFTISRDNAKNTLYLQMNNLKPEDTAIYYCVIAGYSDWGQGTQVTVSS 5E5VH5QLQLVESGGGLVQPGGSLRLSCAASGFTFGTYSMRWVRQVP 16RKALEWVSSIHTDGGGTAYRDSVKGRFTISRDNAKNTLYLQMNNLKPEDTAIYYCVIAGYSDWGQGTQVTVSS 5E5VH12QLQLVESGGGLVQPGGSLRLSCAASGFTFGTYSMRWVRQVP 17RKALEWVSSISTDGGGTAYRDSVKGRFTISRDNAKNTLYLQMNNLKPEDTAIYYCVIHGYSDWGQGTQVTVSS 5E5VH5VQLQLVESGGGLVQPGGSLRLSCAASGFTFGTYSMRWVRQVP 18 H12RKALEWVSSIHTDGGGTAYRDSVKGRFTISRDNAKNTLYLQMNNLKPEDTAIYYCVIHGYSDWGQGTQVTVSS

TABLE 4 VL amino acid sequences of exemplary anti-ApoC3 antibodies.SEQ ID VL clone Amino acid Sequence NO 5E5VLWTATMLTQSPGSLSVVPGESASISCKTSQGLVHSDGKTYFYWFL 19QKPGQSPQQLIYQVSNRASGVPDRFTGSGSGTDFTLKISGVK AEDAGVYYCAQGTYYPHTFGSGTRLEIK5E5VL8 ATMLTQSPGSLSVVPGESASISCKTSQGLVHSDGKTYFYWFL 20QKPGQSPQQLIYQVSNRASGVPDRFTGSGSGTDFTLKISGVK AEDAGVYYCAHGTYYPHTFGSGTRLEIK

TABLE 5 VH and VL sequences of exemplary anti-ApoC3 antibodies. SEQ SEQID ID Antibody VH NO VL NO 5E5WT 5E5VHWT 15 5E5VLWT 19 5E5VH5_VLWT5E5VH5 16 5E5VLWT 19 5E5VH12_VLWT 5E5VH12 17 5E5VLWT 19 5E5VH5VH12_VLWT5E5VH5VH12 18 5E5VLWT 19 5E5VHWT_VL8 5E5VHWT 15 5E5VL8 20 5E5VH5_VL85E5VH5 16 5E5VL8 20 5E5VH12_VL8 5E5VH12 17 5E5VL8 20 5E5VH5VH12_VL85E5VH5VH12 18 5E5VL8 20

TABLE 6Sequences of exemplary heavy chain and light chain constant regions.Constant SEQ Region Amino Acid Sequence ID NO HumanASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA 21 IgG₁LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN constantTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS regionRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN wild-typeSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK HumanASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA 36 IgG₁LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN constantTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS regionRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN wild-typeSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPG HumanASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA 22 IgG₁LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN constantTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL

I

region YTE R

PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK HumanASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA 37 IgG₁LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN constantTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL

I

region YTE R

PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPG HumanASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA 23 IgG₁LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN constantTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS regionRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN HNanceSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE AL

HYTQKSLSLSPGK Human ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA 38IgG₁ LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN constantTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS regionRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN HNanceSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE AL

HYTQKSLSLSPG Human ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA 24 IgG₁LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN constantTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS regionRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN XtendSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV

HE ALH

HYTQKSLSLSPGK Human ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA 39IgG₁ LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN constantTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS regionRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN XtendSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV

HE ALH

HYTQKSLSLSPG Human ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA 40 IgG4LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS constantNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRT regionPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNST wild-typeYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL HNHYTQKSLSLSLGK HumanASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA 41 IgG₄ S228PLTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS constantNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRT regionPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNST wild-typeYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL HNHYTQKSLSLSLG HumanASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA 42 IgG₄ S228PLTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS constantNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTL

I

R

region YTE PEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL HNHYTQKSLSLSLGK HumanASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA 43 IgG₄ S228PLTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS constantNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLYITRE region YTEPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL HNHYTQKSLSLSLG HumanASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA 44 IgG₄ S228PLTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS constantNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRT regionPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNST HNanceYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL

HYTQKSLSLSLGK Human ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA 45IgG₄ S228P LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS constantNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRT regionPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNST HNanceYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL KFHYTQKSLSLSLG HumanASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA 46 IgG₄ S228PLTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS constantNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRT regionPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNST XtendYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSV

HEAL H

HYTQKSLSLSLGK Human ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA 47IgG4₄ S228P LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS constantNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRT regionPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNST XtendYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEAL HSHYTQKSLSLSLG Human IgκRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD 25 constantNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE region VTHQGLSSPVTKSFNRGECHuman Igλ GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKA 26 constantDGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQ region VTHEGSTVEKTVAPTECS

TABLE 7 Full heavy chain and light chain sequences of exemplaryanti-ApoC3 antibodies. Antibody SEQ chain Amino Acid Sequence ID NO5E5VH5 QLQLVESGGGLVQPGGSLRLSCAASGFTFGTYSMRWVRQVPRKA 27LEWVSSIHTDGGGTAYRDSVKGRFTISRDNAKNTLYLQMNNLKPEDTAIYYCVIAGYSDWGQGTQVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 5E5VH5_YTEQLQLVESGGGLVQPGGSLRLSCAASGFTFGTYSMRWVRQVPRKA 28LEWVSSIHTDGGGTAYRDSVKGRFTISRDNAKNTLYLQMNNLKPEDTAIYYCVIAGYSDWGQGTQVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTL

I

R

PEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 5E5VH5_HNanceQLQLVESGGGLVQPGGSLRLSCAASGFTFGTYSMRWVRQVPRKA 29LEWVSSIHTDGGGTAYRDSVKGRFTISRDNAKNTLYLQMNNLKPEDTAIYYCVIAGYSDWGQGTQVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEAL

HYTQKSLSLSPGK 5E5VH5_Xtend QLQLVESGGGLVQPGGSLRLSCAASGFTFGTYSMRWVRQVPRKA30 LEWVSSIHTDGGGTAYRDSVKGRFTISRDNAKNTLYLQMNNLKPEDTAIYYCVIAGYSDWGQGTQVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSV

HEALH

HYTQKSLSLSPGK 5E5VH12 QLQLVESGGGLVQPGGSLRLSCAASGFTFGTYSMRWVRQVPRKA 31LEWVSSISTDGGGTAYRDSVKGRFTISRDNAKNTLYLQMNNLKPEDTAIYYCVIHGYSDWGQGTQVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 5E5VH12_YTEQLQLVESGGGLVQPGGSLRLSCAASGFTFGTYSMRWVRQVPRKA 32LEWVSSISTDGGGTAYRDSVKGRFTISRDNAKNTLYLQMNNLKPEDTAIYYCVIHGYSDWGQGTQVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTL

I

R

PEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 5E5VH12_HNanceQLQLVESGGGLVQPGGSLRLSCAASGFTFGTYSMRWVRQVPRKA 33LEWVSSISTDGGGTAYRDSVKGRFTISRDNAKNTLYLQMNNLKPEDTAIYYCVIHGYSDWGQGTQVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEAL

HYTQKSLSLSPGK 5E5VH12_Xtend QLQLVESGGGLVQPGGSLRLSCAASGFTFGTYSMRWVRQVPRKA34 LEWVSSISTDGGGTAYRDSVKGRFTISRDNAKNTLYLQMNNLKPEDTAIYYCVIHGYSDWGQGTQVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSV

HEALH

HYTQKSLSLSPGK 5E5VL8_full ATMLTQSPGSLSVVPGESASISCKTSQGLVHSDGKTYFYWFLQKP35 light chain GQSPQQLIYQVSNRASGVPDRFTGSGSGTDFTLKISGVKAEDAGVYYCAHGTYYPHTFGSGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to ApoC3 (e.g., human ApoC3), theantibody comprising a VH domain comprising one, two, or all three of theCDRs of a VH domain set forth in Table 3 herein. In certain embodiments,the antibody comprises the CDRH1 of one of VH domains set forth in Table3. In certain embodiments, the antibody comprises the CDRH2 of one ofthe VH domains set forth in Table 3. In certain embodiments, theantibody comprises the CDRH3 of one of the VH domains set forth in Table3.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to ApoC3 (e.g., human ApoC3), theantibody comprising a VL domain comprising one, two, or all three of theCDRs of a VL domain disclosed in Table 4 herein. In certain embodiments,the antibody comprises the CDRL1 of one of VL domains set forth in Table4. In certain embodiments, the antibody comprises the CDRL2 of one ofthe VL domains set forth in Table 4. In certain embodiments, theantibody comprises the CDRL3 of one of the VL domains set forth in Table4.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to ApoC3 (e.g., human ApoC3), theantibody comprising a heavy chain variable region having complementaritydetermining regions CDRH1, CDRH2 and CDRH3, and a light chain variableregion having complementarity determining regions CDRL1, CDRL2 andCDRL3, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 comprise theamino acid sequences of the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3regions, respectively, of an antibody set forth in Table 5.

In certain embodiments, the CDRs of an antibody can be determinedaccording to Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) andKabat et al., Sequences of protein of immunological interest (1991). Incertain embodiments, the light chain CDRs of an antibody are determinedaccording to Kabat and the heavy chain CDRs of an antibody aredetermined according to MacCallum (supra).

In certain embodiments, the CDRs of an antibody can be determinedaccording to the Chothia numbering scheme, which refers to the locationof immunoglobulin structural loops (see, e.g., Chothia C & Lesk A M,(1987), J Mol Biol 196: 901-917; Al-Lazikani B et al., (1997) J Mol Biol273: 927-948; Chothia C et al., (1992) J Mol Biol 227: 799-817;Tramontano A et al., (1990) J Mol Biol 215(1): 175-82; and U.S. Pat. No.7,709,226). Typically, when using the Kabat numbering convention, theChothia CDRH1 loop is present at heavy chain amino acids 26 to 32, 33,or 34, the Chothia CDRH2 loop is present at heavy chain amino acids 52to 56, and the Chothia CDRH3 loop is present at heavy chain amino acids95 to 102, while the Chothia CDRL1 loop is present at light chain aminoacids 24 to 34, the Chothia CDRL2 loop is present at light chain aminoacids 50 to 56, and the Chothia CDRL3 loop is present at light chainamino acids 89 to 97. The end of the Chothia CDRH1 loop when numberedusing the Kabat numbering convention varies between H32 and H34depending on the length of the loop (this is because the Kabat numberingscheme places the insertions at H35A and H35B; if neither 35A nor 35B ispresent, the loop ends at 32; if only 35A is present, the loop ends at33; if both 35A and 35B are present, the loop ends at 34).

In certain embodiments, the CDRs of an antibody can be determinedaccording to the IMGT numbering system as described in Lefranc M-P,(1999) The Immunologist 7: 132-136 and Lefranc M-P et al., (1999)Nucleic Acids Res 27: 209-212. According to the IMGT numbering scheme,CDRH1 is at positions 26 to 35, CDRH2 is at positions 51 to 57, CDRH3 isat positions 93 to 102, CDRL1 is at positions 27 to 32, CDRL2 is atpositions 50 to 52, and CDRL3 is at positions 89 to 97.

In certain embodiments, the CDRs of an antibody can be determinedaccording to the AbM numbering scheme, which refers to AbM hypervariableregions, which represent a compromise between the Kabat CDRs and Chothiastructural loops, and are used by Oxford Molecular's AbM antibodymodeling software (Oxford Molecular Group, Inc.).

In certain embodiments, the CDRs of an antibody can be determinedaccording to MacCallum R M et al., (1996) J Mol Biol 262: 732-745. Seealso, e.g., Martin A. “Protein Sequence and Structure Analysis ofAntibody Variable Domains,” in Antibody Engineering, Kontermann andDübel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001).

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to ApoC3 (e.g., human ApoC3), whereinthe antibody comprises a heavy chain variable region comprising theCDRH1, CDRH2, and CDRH3 region amino acid sequences of a VH domain setforth in Table 3, and a light chain variable region comprising theCDRL1, CDRL2, and CDRL3 region amino acid sequences of a VL domain setforth in Table 4, wherein each CDR is independently defined inaccordance with the Kabat, Chothia, IMGT, MacCallum, or AbM definitionof a CDR, as disclosed herein.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to ApoC3 (e.g., human ApoC3), theantibody comprising a heavy chain variable region having complementaritydetermining regions CDRH1, CDRH2 and CDRH3, and a light chain variableregion having complementarity determining regions CDRL1, CDRL2 andCDRL3, and wherein:

(a) the CDRH1 comprises the amino acid sequence of TYSMR (SEQ ID NO: 3);(b) the CDRH2 comprises the amino acid sequence of SIX₁TDGGGTAYRDSVKG,wherein X₁ is S or H (SEQ ID NO: 4);(c) the CDRH3 comprises the amino acid sequence of X₂GYSD, wherein X₂ isA or H (SEQ ID NO: 5);(d) the CDRL1 comprises the amino acid sequence of KTSQGLVHSDGKTYFY (SEQID NO: 6);(e) the CDRL2 comprises the amino acid sequence of QVSNRAS (SEQ ID NO:7); and/or (f) the CDRL3 comprises the amino acid sequence ofAX₃GTYYPHT, wherein X₃ is Q or H (SEQ ID NO: 8).

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to ApoC3 (e.g., human ApoC3), theantibody comprising a heavy chain variable region having complementaritydetermining regions CDRH1, CDRH2 and CDRH3, and a light chain variableregion having complementarity determining regions CDRL1, CDRL2 andCDRL3, and wherein:

(a) the CDRH1 comprises the amino acid sequence of TYSMR (SEQ ID NO: 3);(b) the CDRH2 comprises the amino acid sequence of SIX₁TDGGGTAYRDSVKG,wherein X₁ is S or H (SEQ ID NO: 4);(c) the CDRH3 comprises the amino acid sequence of X₂GYSD, wherein X₂ isA or H (SEQ ID NO: 5);(d) the CDRL1 comprises the amino acid sequence of KTSQGLVHSDGKTYFY (SEQID NO: 6);(e) the CDRL2 comprises the amino acid sequence of QVSNRAS (SEQ ID NO:7); and(f) the CDRL3 comprises the amino acid sequence of AX₃GTYYPHT, whereinX₃ is Q or H (SEQ ID NO: 8),and wherein at least one of X₁, X₂, and X₃ is H.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to ApoC3 (e.g., human ApoC3), theantibody comprising:

(a) a CDRH1 comprising the amino acid sequence of SEQ ID NO: 3;(b) a CDRH2 comprising the amino acid sequence of SEQ ID NO: 9 or 11;(c) a CDRH3 comprising the amino acid sequence of SEQ ID NO: 10 or 12;(d) a CDRL1 comprising the amino acid sequence of SEQ ID NO: 6;(e) a CDRL2 comprising the amino acid sequence of SEQ ID NO: 7; and/or(f) a CDRL3 comprising the amino acid sequence of SEQ ID NO: 13 or 14.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to ApoC3 (e.g., human ApoC3), theantibody comprising:

(a) a CDRH1 comprising the amino acid sequence of SEQ ID NO: 3;(b) a CDRH2 comprising the amino acid sequence of SEQ ID NO: 9 or 11;(c) a CDRH3 comprising the amino acid sequence of SEQ ID NO: 10 or 12;(d) a CDRL1 comprising the amino acid sequence of SEQ ID NO: 6;(e) a CDRL2 comprising the amino acid sequence of SEQ ID NO: 7; and(f) a CDRL3 comprising the amino acid sequence of SEQ ID NO: 13 or 14,and wherein the isolated antibody does not comprise the CDRH1, CDRH2,CDRH3, CDRL1, CDRL2, and CDRL3 sequences set forth in SEQ ID NOs: 3, 9,10, 6, 7, and 13, respectively.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to ApoC3 (e.g., human ApoC3), whereinthe antibody comprises a VH domain comprising the CDRH1, CDRH2 and CDRH3amino acid sequences set forth in SEQ ID NOs: 3, 9, and 10; 3, 11, and10; 3, 9, and 12; or 3, 11, and 12, respectively. In certainembodiments, the VH domain comprises the CDRH1, CDRH2 and CDRH3 aminoacid sequences set forth in SEQ ID NOs: 3, 11, and 10, respectively. Incertain embodiments, the VH domain comprises the CDRH1, CDRH2 and CDRH3amino acid sequences set forth in SEQ ID NOs: 3, 9, and 12,respectively. In certain embodiments, the VH domain comprises the CDRH1,CDRH2 and CDRH3 amino acid sequences set forth in SEQ ID NOs: 3, 11, and12, respectively.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to ApoC3 (e.g., human ApoC3), whereinthe antibody comprises a VL domain comprising the CDRL1, CDRL2 and CDRL3amino acid sequences set forth in SEQ ID NOs: 6, 7, and 13; or 6, 7, and14, respectively. In certain embodiments, the VL domain comprises theCDRL1, CDRL2 and CDRL3 amino acid sequences set forth in SEQ ID NOs: 6,7, and 14, respectively.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to ApoC3 (e.g., human ApoC3), whereinthe antibody comprises a heavy chain variable region comprising CDRH1,CDRH2, and CDRH3 regions, and a light chain variable region comprisingCDRL1, CDRL2, and CDRL3 regions, wherein the CDRH1, CDRH2, CDRH3, CDRL1,CDRL2, and CDRL3 regions comprise the amino acid sequences set forth inSEQ ID NOs: 3, 11, 10, 6, 7, and 13; 3, 9, 12, 6, 7, and 13; 3, 9, 10,6, 7, and 14; 3, 11, 10, 6, 7, and 14; 3, 9, 12, 6, 7, and 14; 3, 11,12, 6, 7, and 13; or 3, 11, 12, 6, 7, and 13, respectively. In certainembodiments, the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 regionscomprise the amino acid sequences set forth in SEQ ID NOs: 3, 11, 10, 6,7, and 14, respectively. In certain embodiments, the CDRH1, CDRH2,CDRH3, CDRL1, CDRL2, and CDRL3 regions comprise the amino acid sequencesset forth in SEQ ID NOs: 3, 9, 12, 6, 7, and 14, respectively.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to ApoC3 (e.g., human ApoC3),comprising a heavy chain variable region comprising an amino acidsequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., atleast 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%)identical to the amino acid sequence set forth in SEQ ID NO: 15, 16, 17,or 18. In certain embodiments, the antibody comprises a heavy chainvariable region having the amino acid sequence set forth in SEQ ID NO:15, 16, 17, or 18. In certain embodiments, the antibody comprises aheavy chain variable region having the amino acid sequence set forth inSEQ ID NO: 16. In certain embodiments, the antibody comprises a heavychain variable region having the amino acid sequence set forth in SEQ IDNO: 17. In certain embodiments, the antibody comprises a heavy chainvariable region having the amino acid sequence set forth in SEQ ID NO:18.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to ApoC3 (e.g., human ApoC3),comprising a light chain variable region comprising an amino acidsequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., atleast 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%)identical to the amino acid sequence set forth in SEQ ID NO: 19 or 20.In certain embodiments, the antibody comprises a light chain variableregion having the amino acid sequence set forth in SEQ ID NO: 20.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to ApoC3 (e.g., human ApoC3),comprising a heavy chain variable region comprising an amino acidsequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., atleast 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%)identical to the amino acid sequence set forth in SEQ ID NO: 15, 16, 17,or 18, and alight chain variable region comprising an amino acidsequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., atleast 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%)identical to the amino acid sequence set forth in SEQ ID NO: 19 or 20.In certain embodiments, the antibody comprises a heavy chain variableregion having the amino acid sequence set forth in SEQ ID NO: 15, 16,17, or 18, and a light chain variable region having the amino acidsequence set forth in SEQ ID NO: 19 or 20. In certain embodiments, theantibody comprises a heavy chain variable region and light chainvariable region having the amino acid sequences set forth in SEQ ID NOs:16 and 19, 17 and 19, 18 and 19, 15 and 20, 16 and 20, 17 and 20, or 18and 20, respectively. In certain embodiments, the antibody comprises aheavy chain variable region and light chain variable region having theamino acid sequences set forth in SEQ ID NO: 16 and 20, respectively. Incertain embodiments, the antibody comprises a heavy chain variableregion and light chain variable region having the amino acid sequencesset forth in SEQ ID NO: 17 and 20, respectively.

Any Ig constant region can be used in the isolated antibodies disclosedherein. In certain embodiments, the Ig constant region is a constantregion of human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin (Ig)molecule, and/or a constant region of any class (e.g., IgG₁, IgG₂, IgG₃,IgG₄, IgA₁, and IgA₂) or any subclass (e.g., IgG₂a and IgG₂b) ofimmunoglobulin molecule. In certain embodiments, the Ig constant regionis a human or humanized Ig constant region.

In certain embodiments, the constant region is a variant of a wild typehuman Ig (e.g., IgG) heavy chain constant region, and wherein thevariant human Ig heavy chain constant region has an increased affinity(e.g., increased by at least 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 15,or 20 fold) for human neonatal Fc receptor (FcRn) at acidic pH (e.g., pH5.5 to pH 6) relative to the affinity of the corresponding wild typehuman Ig heavy chain constant region for human FcRn under the sameconditions. In certain embodiments, the variant human Ig heavy chainconstant region has a similar or decreased affinity (e.g., increased byno more than 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 fold,equal to, or decreased) for human neonatal Fc receptor (FcRn) atphysiological pH (e.g., at pH 7.4) relative to the affinity of the wildtype human Ig heavy chain constant region for human FcRn under the sameconditions. In certain embodiments, the constant region comprises one,two, or more amino acids (e.g., having one or more substitutions,insertions or deletions) from a wild-type Ig (e.g., IgG) constant domainor FcRn-binding fragment thereof (e.g., an Fc or hinge-Fc domainfragment). In certain embodiments, the half-life of the antibody withthe variant constant region in vivo is increased relative to thehalf-life of the corresponding antibody with the wild-type constantdomain or FcRn-binding fragment thereof in vivo. See, e.g.,International Publication Nos. WO 02/060919; WO 98/23289; and WO97/34631; and U.S. Pat. Nos. 5,869,046, 6,121,022, 6,277,375, 6,165,745,8,088,376, and 8,163,881, all of which are herein incorporated byreference in their entireties, for examples of mutations that willincrease the half-life of an antibody in vivo. In certain embodiment,the one or more different amino acid are in the second constant (CH2)domain (residues 231-340 of human IgG₁) and/or the third constant (CH3)domain (residues 341-447 of human IgG₁), numbered according to the EUnumbering system. In certain embodiments, the constant region of the IgG(e.g., IgG₁, IgG₂, or IgG₄) of an antibody disclosed herein comprisesthe amino acids tyrosine (Y) threonine (T), and glutamic acid (E) atpositions 252, 254, and 256, respectively, numbered according to the EUnumbering system. See U.S. Pat. No. 7,658,921, which is hereinincorporated by reference in its entirety. This type of IgG, referred toas “YTE IgG” has been shown to display fourfold increased half-life ascompared to wild-type versions of the same antibody (see Dall'Acqua W Fet al., (2006) J Biol Chem 281: 23514-24, which is herein incorporatedby reference in its entirety). In certain embodiments, the constantregion of the IgG (e.g., IgG₁) of an antibody disclosed herein comprisesthe amino acid alanine (A), serine (S), tyrosine (Y), or phenylalanine(F) at position 434, numbered according to the EU numbering system. Incertain embodiments, the constant region of the IgG (e.g., IgG₁, IgG₂,or IgG₄) of an antibody disclosed herein comprises the amino acidslysine (K), phenylalanine (F), and tyrosine (Y) at positions 433, 434,and 436, respectively, numbered according to the EU numbering system. Incertain embodiments, the constant region of the IgG (e.g., IgG₁, IgG₂,or IgG₄) of an antibody disclosed herein comprises the amino acidsleucine (L) and serine (S) at positions 428 and 434, respectively,numbered according to the EU numbering system. Additional IgG constantregions that may have increased affinity to FcRn under acidic conditionare described in Ward et al., Mol. Immunol. (2015) 67(200):131-41, whichis herein incorporated by reference in its entirety. In certainembodiments, an antibody comprises an IgG constant domain comprisingone, two, three or more amino acid substitutions of amino acid residuesat positions 251-257, 285-290, 308-314, 385-389, and 428-436, numberedaccording to the EU numbering system. In certain embodiments, theisolated antibodies disclosed herein comprise a heavy chain constantregion comprising the amino acid sequence set forth in SEQ ID NO: 21,22, 23, 24, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, or 47. Incertain embodiments, the isolated antibodies disclosed herein comprise alight chain constant region comprising the amino acid sequence set forthin SEQ ID NO: 25 or 26.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to ApoC3 (e.g., human ApoC3),comprising a heavy chain comprising the amino acid sequence set forth inSEQ ID NO: 27, 28, 29, 30, 31, 32, 33, or 34. In certain embodiments,the instant disclosure provides an isolated antibody that specificallybinds to ApoC3 (e.g., human ApoC3), comprising a light chain comprisingthe amino acid sequence set forth in SEQ ID NO: 35. In certainembodiments, the instant disclosure provides an isolated antibody thatspecifically binds to ApoC3 (e.g., human ApoC3), comprising a heavychain and a light chain comprising the amino acid sequences set forth inSEQ ID NOs: 27 and 35, 28 and 35, 29 and 35, 30 and 35, 31 and 35, 32and 35, 33 and 35, or 34 and 35, respectively.

3. Methods of Use

ApoC3 inhibits TRL (e.g., VLDL) and TRL remnant uptake and clearance byhepatocytes and inhibits lipoprotein lipase-mediated lipolysis of TRL(e.g., VLDL), thereby functioning to increase triglyceride levels in theblood of a subject. In certain embodiments, the anti-ApoC3 antibodiesdisclosed herein can attenuate the ability of ApoC3 to inhibit TRL(e.g., VLDL) and TRL remnant uptake and clearance by hepatocytes orattenuate the ability of ApoC3 to inhibit lipoprotein lipase-mediatedlipolysis of TRL (e.g., VLDL). Accordingly, in certain embodiments, theinstant disclosure provides a method for inhibiting the activity ofApoC3 in the blood of a subject, the method comprising administering tothe subject an effective amount of an anti-ApoC3 antibody orpharmaceutical composition disclosed herein. In certain embodiments, theactivity of ApoC3 is inhibition of TRL (e.g., VLDL) and TRL remnantsuptake and clearance by hepatocytes. In certain embodiments, theactivity of ApoC3 is inhibition of lipoprotein lipase-mediated lipolysisof TRL. In certain embodiments, the activity of ApoC3 is inhibition ofTRL (e.g., VLDL) and TRL remnants uptake and clearance by hepatocytesand inhibition of lipoprotein lipase-mediated lipolysis of TRL.

The anti-ApoC3 antibodies disclosed herein are useful for increasing therate of clearance of ApoC3 and/or ApoB (e.g., ApoB48 and/or ApoB100)from the blood in a subject. Accordingly, in certain embodiments, theinstant disclosure provides a method for increasing the rate ofclearance of ApoC3 and/or ApoB (e.g., ApoB48 and/or ApoB100) from theblood in a subject, the method comprising administering to the subjectan effective amount of an anti-ApoC3 antibody or pharmaceuticalcomposition disclosed herein.

The anti-ApoC3 antibodies disclosed herein are useful for reducing thelevel of ApoC3 and/or ApoB (e.g., ApoB48 and/or ApoB100) in the blood ofa subject. Accordingly, in certain embodiments, the instant disclosureprovides a method for reducing the level of ApoC3 and/or ApoB (e.g.,ApoB48 and/or ApoB100) in the blood of a subject, the method comprisingadministering to the subject an effective amount of an anti-ApoC3antibody or pharmaceutical composition disclosed herein. In certainembodiments, the method reduces the level of ApoC3 and/or ApoB (e.g.,ApoB48 and/or ApoB100) in the blood of a subject by at least 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 98%, or 99%, as assessed by methods disclosed herein orby methods known to one of skill in the art. In certain embodiments, themethod reduces the levels of ApoC3 and/or ApoB (e.g., ApoB48 and/orApoB100) in the blood in a subject by at least about 1.1 fold, 1.2 fold,1.3 fold, 1.4 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90fold, or 100 fold, as assessed by methods disclosed herein or by methodsknown to one of skill in the art. In certain embodiments, the reductionin the levels of ApoC3 and/or ApoB (e.g., ApoB48 and/or ApoB100) in theblood in the subject is maintained for at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 days, or at least 1, 2, 3, 4,5, 6, 7, or 8 weeks.

The anti-ApoC3 antibodies disclosed herein are useful for reducingtriglyceride levels in the blood of a subject. Accordingly, in certainembodiments, the instant disclosure provides a method for reducingtriglyceride levels in the blood of a subject, the method comprisingadministering to the subject an effective amount of an anti-ApoC3antibody or pharmaceutical composition disclosed herein.

The anti-ApoC3 antibodies disclosed herein are useful for the treatmentof hypertriglyceridemia. Accordingly, in certain embodiments, theinstant disclosure provides a method for treating hypertriglyceridemiain a subject, the method comprising administering to the subject aneffective amount of an anti-ApoC3 antibody or pharmaceutical compositiondisclosed herein. In certain embodiments, the instant disclosureprovides a method for treating chylomicronemia in a subject, the methodcomprising administering to the subject an effective amount of ananti-ApoC3 antibody or pharmaceutical composition disclosed herein. Incertain embodiments, the instant disclosure provides a method fortreating chylomicronemia syndrome in a subject, the method comprisingadministering to the subject an effective amount of an anti-ApoC3antibody or pharmaceutical composition disclosed herein.

The anti-ApoC3 antibodies disclosed herein are useful for the treatmentand prevention of post-prandial lipemia in a subject. Accordingly, incertain embodiments, the instant disclosure provides a method forinhibiting post-prandial lipemia in a subject, the method comprisingadministering to the subject an effective amount of an anti-ApoC3antibody or pharmaceutical composition disclosed herein. The anti-ApoC3antibody can be administered to the subject prior to, during, or after ameal.

Without wishing to be bound by theory, Applicants believe that, incertain embodiments, the antibodies disclosed herein are capable ofreducing the levels of post-prandial chylomicron or chylomicron remnantsin a subject when administered to the subject prior to, during, or aftera meal. Accordingly, in certain embodiments, the instant disclosureprovides a method for reducing the levels of post-prandial chylomicronor chylomicron remnants in a subject, the method comprisingadministering to the subject an effective amount of an anti-ApoC3antibody or pharmaceutical composition disclosed herein. The anti-ApoC3antibody can be administered to the subject prior to, during, or after ameal.

The reduction of triglyceride levels in blood in patients withhypertriglyceridemia may reduce the risk of development of pancreatitis.Accordingly, in certain embodiments, the instant disclosure provides amethod for reducing the risk of pancreatitis in a subject withhypertriglyceridemia, the method comprising administering to the subjectan effective amount of an anti-ApoC3 antibody or pharmaceuticalcomposition disclosed herein

The anti-ApoC3 antibodies disclosed herein are useful for reducing therisk of cardiovascular disease in a subject. Accordingly, in certainembodiments, the instant disclosure provides a method for reducing therisk of cardiovascular disease in a subject with hypertriglyceridemia,the method comprising administering to the subject an effective amountof an anti-ApoC3 antibody or pharmaceutical composition disclosedherein. The risk of developing any cardiovascular disease associatedwith or caused by hypertriglyceridemia or excessive post prandiallipemia can be reduced by administration of an anti-ApoC3 antibody orpharmaceutical composition disclosed herein. Cardiovascular disease forwhich the risk can be reduced include without limitation coronary arterydisease, atherosclerosis, angina, myocardial infarction, and stroke.

The anti-ApoC3 antibodies or pharmaceutical compositions disclosedherein can be administered either alone or in combination an additionaltherapeutic agent. In certain embodiments, the additional therapeuticagent is another lipid lowering agent. Any one or more lipid loweringagent can be used in combination with an anti-ApoC3 antibody orpharmaceutical composition disclosed herein. Suitable lipid loweringagents include without limitation HMG-CoA reductase inhibitors (e.g.,atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin,rosuvastatin or simvastatin), fibrates, niacin, bile acid sequestrants(e.g., cholestyramine, colestipol, and colesevelam), inhibitors ofdietary cholesterol absorption (e.g., ezetimibe), microsomaltriglyceride transfer protein (MTP) inhibitors (e.g., lomitapide),phytosterols, pancreatic lipase inhibitors (e.g., orlistat), cholesterylester transfer protein inhibitors, squalene synthase inhibitors (e.g.,TAK-475, zaragozic acid, and RPR 107393), ApoA-1 Milano, succinobucol(AGI-1067), Apoprotein-B inhibitors (e.g., Mipomersen), and proproteinconvertase subtilisin/kexin type 9 (PCSK9) inhibitors (e.g., alirocumab,evolocumab, and bococizumab). In certain embodiments, the additionallipid lowering agent is a combination of ezetimibe and an HMG-CoAreductase inhibitor. In certain embodiments, the lipid lowering agent isa combination of ezetimibe, an HMG-CoA reductase inhibitor, and a PCSK9inhibitor.

The anti-ApoC3 antibodies or pharmaceutical compositions disclosedherein may be delivered to a subject by a variety of routes. Theseinclude, but are not limited to, parenteral, intradermal, intramuscular,intraperitoneal, intravenous, and subcutaneous routes. In certainembodiments, the antibody or pharmaceutical composition disclosed hereinis delivered subcutaneously or intravenously.

The amount of an anti-ApoC3 antibody or pharmaceutical compositiondisclosed herein which will be effective in the treatment or preventionof a condition will depend on the nature of the disease, and can beempirically determined by standard clinical techniques. The precise doseto be employed in a composition will also depend on the route ofadministration, and the seriousness of the infection or disease causedby it, and should be decided according to the judgment of thepractitioner and each subject's circumstances. For example, effectivedoses may also vary depending upon means of administration, target site,physiological state of the patient (including age, body weight andhealth), whether the patient is human or an animal, other medicationsadministered, or whether treatment is prophylactic or therapeutic. Theanti-ApoC3 antibodies or pharmaceutical compositions disclosed hereincan be administered at any frequency (e.g., about every week, every twoweeks, every three weeks, every four weeks, every month, or every twomonths). Usually, the patient is a human, but non-human mammalsincluding transgenic mammals can also be treated. Treatment dosages andregimens are optimally titrated to optimize safety and efficacy.

The anti-ApoC3 antibodies disclosed herein can also be used to assayApoC3 (e.g., human ApoC3) protein levels in a biological sample usingclassical immunohistological methods known to those of skill in the art,including immunoassays, such as the enzyme linked immunosorbent assay(ELISA), immunoprecipitation, or Western blotting. Suitable antibodyassay labels are known in the art and include enzyme labels, such as,glucose oxidase; radioisotopes, such as iodine (¹²⁵I, ¹²¹I) carbon(¹⁴C), sulfur (³⁵S), tritium (³H), indium (¹²¹In), and technetium(⁹⁹Tc); luminescent labels, such as luminol; and fluorescent labels,such as fluorescein and rhodamine, and biotin. Such labels can be usedto label an antibody disclosed herein. Alternatively, a second antibodythat recognizes an anti-ApoC3 antibody disclosed herein can be labeledand used in combination with an anti-ApoC3 antibody to detect ApoC3(e.g., human ApoC3) protein levels.

Assaying for the expression level of ApoC3 (e.g., human ApoC3) proteinis intended to include qualitatively or quantitatively measuring orestimating the level of ApoC3 (e.g., human ApoC3) protein in a firstbiological sample either directly (e.g., by determining or estimatingabsolute protein level) or relatively (e.g., by comparing to the diseaseassociated protein level in a second biological sample). ApoC3 (e.g.,human ApoC3) polypeptide expression level in the first biological samplecan be measured or estimated and compared to a standard ApoC3 (e.g.,human ApoC3) protein level, the standard being taken from a secondbiological sample obtained from an individual not having the disorder orbeing determined by averaging levels from a population of individualsnot having the disorder. As will be appreciated in the art, once the“standard” ApoC3 (e.g., human ApoC3) polypeptide level is known, it canbe used repeatedly as a standard for comparison.

As used herein, the term “biological sample” refers to any biologicalsample obtained from a subject, cell line, tissue, or other source ofcells potentially expressing ApoC3 (e.g., human ApoC3). Methods forobtaining tissue biopsies and body fluids from animals (e.g., humans)are well known in the art. Biological samples include peripheralmononuclear blood cells.

The anti-ApoC3 antibodies disclosed herein can be used for prognostic,diagnostic, monitoring and screening applications, including in vitroand in vivo applications well known and standard to the skilled artisanand based on the present description. Prognostic, diagnostic, monitoringand screening assays and kits for in vitro assessment and evaluation ofimmune system status or immune response may be utilized to predict,diagnose and monitor to evaluate patient samples including those knownto have or suspected of having elevated ApoC3 activity. In oneembodiment, an anti-ApoC3 antibody can be used in immunohistochemistryof biopsy samples. In another embodiment, an anti-ApoC3 antibody can beused to detect levels of ApoC3 (e.g., human ApoC3), which levels canthen be linked to certain disease symptoms. Anti-ApoC3 antibodiesdisclosed herein may carry a detectable or functional label. Whenfluorescence labels are used, currently available microscopy andfluorescence-activated cell sorter analysis (FACS) or combination ofboth methods procedures known in the art may be utilized to identify andto quantitate the specific binding members. Anti-ApoC3 antibodiesdisclosed herein may carry a fluorescence label. Exemplary fluorescencelabels include, for example, reactive and conjugated probes e.g.Aminocoumarin, Fluorescein and Texas red, Alexa Fluor dyes, Cy dyes andDyLight dyes. An anti-ApoC3 antibody may carry a radioactive label, suchas the isotopes ³H, ¹⁴C, ³²P, ³⁵S, ³⁶Cl, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁶⁷Cu, ⁹⁰Y,⁹⁹Tc, ¹¹¹In, ¹¹⁷Lu, ¹²¹I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁹⁸Au, ²¹¹At, ²¹³Bi, ²²⁵Acand ¹⁸⁶Re. When radioactive labels are used, currently availablecounting procedures known in the art may be utilized to identify andquantitate the specific binding of anti-ApoC3 antibody to ApoC3 (e.g.,human ApoC3). In the instance where the label is an enzyme, detectionmay be accomplished by any of the presently utilized colorimetric,spectrophotometric, fluorospectrophotometric, amperometric or gasometrictechniques as known in the art. This can be achieved by contacting asample or a control sample with an anti-ApoC3 antibody under conditionsthat allow for the formation of a complex between the antibody and ApoC3(e.g., human ApoC3). Any complexes formed between the antibody and ApoC3(e.g., human ApoC3) are detected and compared in the sample and thecontrol. The antibodies disclosed herein can also be used to purifyApoC3 (e.g., human ApoC3) via immunoaffinity purification. Also includedherein is an assay system which may be prepared in the form of a testkit for the quantitative analysis of the extent of the presence of, forinstance, ApoC3 (e.g., human ApoC3). The system or test kit may comprisea labeled component, e.g., a labeled ApoC3 antibody, and one or moreadditional immunochemical reagents.

4. Pharmaceutical Compositions

Provided herein are pharmaceutical compositions comprising an anti-ApoC3antibody disclosed herein having the desired degree of purity in aphysiologically acceptable carrier, excipient or stabilizer (Remington'sPharmaceutical Sciences (1990) Mack Publishing Co., Easton, Pa.).Acceptable carriers, excipients, or stabilizers are nontoxic torecipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

In a specific embodiment, pharmaceutical compositions comprise ananti-ApoC3 antibody disclosed herein, and optionally one or moreadditional prophylactic or therapeutic agents, in a pharmaceuticallyacceptable carrier. In a specific embodiment, pharmaceuticalcompositions comprise an effective amount of an antibody disclosedherein, and optionally one or more additional prophylactic ortherapeutic agents, in a pharmaceutically acceptable carrier. In someembodiments, the antibody is the only active ingredient included in thepharmaceutical composition. Pharmaceutical compositions disclosed hereincan be useful in inhibiting, ApoC3 activity and treating a condition,such as cancer or an infectious disease.

Pharmaceutically acceptable carriers used in parenteral preparationsinclude aqueous vehicles, nonaqueous vehicles, antimicrobial agents,isotonic agents, buffers, antioxidants, local anesthetics, suspendingand dispersing agents, emulsifying agents, sequestering or chelatingagents and other pharmaceutically acceptable substances. Examples ofaqueous vehicles include Sodium Chloride Injection, Ringers Injection,Isotonic Dextrose Injection, Sterile Water Injection, Dextrose andLactated Ringers Injection. Nonaqueous parenteral vehicles include fixedoils of vegetable origin, cottonseed oil, corn oil, sesame oil andpeanut oil. Antimicrobial agents in bacteriostatic or fungistaticconcentrations can be added to parenteral preparations packaged inmultiple-dose containers which include phenols or cresols, mercurials,benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acidesters, thimerosal, benzalkonium chloride and benzethonium chloride.Isotonic agents include sodium chloride and dextrose. Buffers includephosphate and citrate. Antioxidants include sodium bisulfate. Localanesthetics include procaine hydrochloride. Suspending and dispersingagents include sodium carboxymethylcelluose, hydroxypropylmethylcellulose and polyvinylpyrrolidone. Emulsifying agents includePolysorbate 80 (TWEEN® 80). A sequestering or chelating agent of metalions includes EDTA. Pharmaceutical carriers also include ethyl alcohol,polyethylene glycol and propylene glycol for water miscible vehicles;and sodium hydroxide, hydrochloric acid, citric acid or lactic acid forpH adjustment.

A pharmaceutical composition may be formulated for any route ofadministration to a subject. Specific examples of routes ofadministration include intranasal, oral, pulmonary, transdermal,intradermal, and parenteral. Parenteral administration, characterized byeither subcutaneous, intramuscular or intravenous injection, is alsocontemplated herein. Injectables can be prepared in conventional forms,either as liquid solutions or suspensions, solid forms suitable forsolution or suspension in liquid prior to injection, or as emulsions.The injectables, solutions and emulsions also contain one or moreexcipients. Suitable excipients are, for example, water, saline,dextrose, glycerol or ethanol. In addition, if desired, thepharmaceutical compositions to be administered can also contain minoramounts of non-toxic auxiliary substances such as wetting or emulsifyingagents, pH buffering agents, stabilizers, solubility enhancers, andother such agents, such as for example, sodium acetate, sorbitanmonolaurate, triethanolamine oleate and cyclodextrins.

Preparations for parenteral administration of an antibody includesterile solutions ready for injection, sterile dry soluble products,such as lyophilized powders, ready to be combined with a solvent justprior to use, including hypodermic tablets, sterile suspensions readyfor injection, sterile dry insoluble products ready to be combined witha vehicle just prior to use and sterile emulsions. The solutions may beeither aqueous or nonaqueous.

If administered intravenously, suitable carriers include physiologicalsaline or phosphate buffered saline (PBS), and solutions containingthickening and solubilizing agents, such as glucose, polyethyleneglycol, and polypropylene glycol and mixtures thereof.

Topical mixtures comprising an antibody are prepared as described forthe local and systemic administration. The resulting mixture can be asolution, suspension, emulsions or the like and can be formulated ascreams, gels, ointments, emulsions, solutions, elixirs, lotions,suspensions, tinctures, pastes, foams, aerosols, irrigations, sprays,suppositories, bandages, dermal patches or any other formulationssuitable for topical administration.

An anti-ApoC3 antibody disclosed herein can be formulated as an aerosolfor topical application, such as by inhalation (see, e.g., U.S. Pat.Nos. 4,044,126, 4,414,209 and 4,364,923, which describe aerosols fordelivery of a steroid useful for treatment of inflammatory diseases,particularly asthma). These formulations for administration to therespiratory tract can be in the form of an aerosol or solution for anebulizer, or as a microfine powder for insufflations, alone or incombination with an inert carrier such as lactose. In such a case, theparticles of the formulation will, in one embodiment, have diameters ofless than 50 microns, in one embodiment less than 10 microns.

An anti-ApoC3 antibody disclosed herein can be formulated for local ortopical application, such as for topical application to the skin andmucous membranes, such as in the eye, in the form of gels, creams, andlotions and for application to the eye or for intracisternal orintraspinal application. Topical administration is contemplated fortransdermal delivery and also for administration to the eyes or mucosa,or for inhalation therapies. Nasal solutions of the antibody alone or incombination with other pharmaceutically acceptable excipients can alsobe administered.

Transdermal patches, including iontophoretic and electrophoreticdevices, are well known to those of skill in the art, and can be used toadminister an antibody. For example, such patches are disclosed in U.S.Pat. Nos. 6,267,983, 6,261,595, 6,256,533, 6,167,301, 6,024,975,6,010715, 5,985,317, 5,983,134, 5,948,433, and 5,860,957.

In certain embodiments, a pharmaceutical composition comprising andisclosed herein is a lyophilized powder, which can be reconstituted foradministration as solutions, emulsions and other mixtures. It may alsobe reconstituted and formulated as solids or gels. The lyophilizedpowder is prepared by dissolving an antibody disclosed herein, or apharmaceutically acceptable derivative thereof, in a suitable solvent.In some embodiments, the lyophilized powder is sterile. The solvent maycontain an excipient which improves the stability or otherpharmacological component of the powder or reconstituted solution,prepared from the powder. Excipients that may be used include, but arenot limited to, dextrose, sorbitol, fructose, corn syrup, xylitol,glycerin, glucose, sucrose or other suitable agent. The solvent may alsocontain a buffer, such as citrate, sodium or potassium phosphate orother such buffer known to those of skill in the art at, in oneembodiment, about neutral pH. Subsequent sterile filtration of thesolution followed by lyophilization under standard conditions known tothose of skill in the art provides the desired formulation. In oneembodiment, the resulting solution will be apportioned into vials forlyophilization. Each vial will contain a single dosage or multipledosages of the compound. The lyophilized powder can be stored underappropriate conditions, such as at about 4° C. to room temperature.Reconstitution of this lyophilized powder with water for injectionprovides a formulation for use in parenteral administration. Forreconstitution, the lyophilized powder is added to sterile water orother suitable carrier. The precise amount depends upon the selectedcompound. Such amount can be empirically determined.

The anti-ApoC3 antibodies disclosed herein and other compositionsprovided herein can also be formulated to be targeted to a particulartissue, receptor, or other area of the body of the subject to betreated. Many such targeting methods are well known to those of skill inthe art. All such targeting methods are contemplated herein for use inthe instant compositions. For non-limiting examples of targetingmethods, see, e.g., U.S. Pat. Nos. 6,316,652, 6,274,552, 6,271,359,6,253,872, 6,139,865, 6,131,570, 6,120,751, 6,071,495, 6,060,082,6,048,736, 6,039,975, 6,004,534, 5,985,307, 5,972,366, 5,900,252,5,840,674, 5,759,542 and 5,709,874.

The compositions to be used for in vivo administration can be sterile.This is readily accomplished by filtration through, e.g., sterilefiltration membranes.

5. Polynucleotides, Vectors and Methods of Producing Anti-ApoC3Antibodies

In another aspect, provided herein are polynucleotides comprising anucleotide sequence encoding an anti-ApoC3 antibody disclosed herein(e.g., a light chain variable region or heavy chain variable region),and vectors, e.g., vectors comprising such polynucleotides forrecombinant expression in host cells (e.g., E. coli and mammaliancells).

As used herein, an “isolated” polynucleotide or nucleic acid molecule isone which is separated from other nucleic acid molecules which arepresent in the natural source (e.g., in a mouse or a human) of thenucleic acid molecule. Moreover, an “isolated” nucleic acid molecule,such as a cDNA molecule, can be substantially free of other cellularmaterial, or culture medium when produced by recombinant techniques, orsubstantially free of chemical precursors or other chemicals whenchemically synthesized. For example, the language “substantially free”includes preparations of polynucleotide or nucleic acid molecule havingless than about 15%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (in particular lessthan about 10%) of other material, e.g., cellular material, culturemedium, other nucleic acid molecules, chemical precursors or otherchemicals. In a specific embodiment, a nucleic acid molecule(s) encodingan antibody disclosed herein is isolated or purified.

In particular aspects, provided herein are polynucleotides comprisingnucleotide sequences encoding antibodies, which specifically bind toApoC3 (e.g., human ApoC3) polypeptide and comprises an amino acidsequence as disclosed herein, as well as antibodies which compete withsuch antibodies for binding to ApoC3 (e.g., human ApoC3) polypeptide(e.g., in a dose-dependent manner), or which binds to the same epitopeas that of such antibodies.

In certain aspects, provided herein are polynucleotides comprising anucleotide sequence encoding the light chain or heavy chain of anantibody disclosed herein. The polynucleotides can comprise nucleotidesequences encoding the VH, VL or CDRs of antibodies disclosed herein(see, e.g., Tables 1-4 herein).

Also provided herein are polynucleotides encoding an anti-ApoC3 antibodythat are optimized, e.g., by codon/RNA optimization, replacement withheterologous signal sequences, and elimination of mRNA instabilityelements. Methods to generate optimized nucleic acids encoding ananti-ApoC3 antibody (e.g., light chain, heavy chain, VH domain, or VLdomain) for recombinant expression by introducing codon changes oreliminating inhibitory regions in the mRNA can be carried out byadapting the optimization methods described in, e.g., U.S. Pat. Nos.5,965,726; 6,174,666; 6,291,664; 6,414,132; and 6,794,498, accordingly.For example, potential splice sites and instability elements (e.g., A/Tor A/U rich elements) within the RNA can be mutated without altering theamino acids encoded by the nucleic acid sequences to increase stabilityof the RNA for recombinant expression. The alterations utilize thedegeneracy of the genetic code, e.g., using an alternative codon for anidentical amino acid. In some embodiments, it can be desirable to alterone or more codons to encode a conservative mutation, e.g., a similaramino acid with similar chemical structure and properties or function asthe original amino acid. Such methods can increase expression of ananti-ApoC3 by at least 1 fold, 2 fold, 3 fold, 4 fold, 5 fold, 10 fold,20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold,or 100 fold or more relative to the expression of an anti-ApoC3 antibodyencoded by polynucleotides that have not been optimized.

In certain embodiments, an optimized polynucleotide sequence encoding ananti-ApoC3 antibody disclosed herein (e.g., VL domain or VH domain) canhybridize to an antisense (e.g., complementary) polynucleotide of anunoptimized polynucleotide sequence encoding an anti-ApoC3 antibodydisclosed herein (e.g., VL domain or VH domain). In specificembodiments, an optimized nucleotide sequence encoding an anti-ApoC3antibody disclosed herein or a fragment hybridizes under high stringencyconditions to antisense polynucleotide of an unoptimized polynucleotidesequence encoding an anti-ApoC3 antibody disclosed herein. In a specificembodiment, an optimized nucleotide sequence encoding an anti-ApoC3antibody disclosed herein hybridizes under high stringency, intermediateor lower stringency hybridization conditions to an antisensepolynucleotide of an unoptimized nucleotide sequence encoding ananti-ApoC3 antibody disclosed herein. Information regardinghybridization conditions has been described, see, e.g., U.S. PatentApplication Publication No. US 2005/0048549 (e.g., paragraphs 72-73),which is incorporated herein by reference.

The polynucleotides can be obtained, and the nucleotide sequence of thepolynucleotides determined, by any method known in the art. Nucleotidesequences encoding antibodies disclosed herein, e.g., antibodiesdescribed in Table 1, and modified versions of these antibodies can bedetermined using methods well known in the art, i.e., nucleotide codonsknown to encode particular amino acids are assembled in such a way togenerate a nucleic acid that encodes the antibody. Such a polynucleotideencoding the antibody can be assembled from chemically synthesizedoligonucleotides (e.g., as described in Kutmeier G et al., (1994),BioTechniques 17: 242-6), which, briefly, involves the synthesis ofoverlapping oligonucleotides containing portions of the sequenceencoding the antibody, annealing and ligating of those oligonucleotides,and then amplification of the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody disclosed hereincan be generated from nucleic acid from a suitable source (e.g., ahybridoma) using methods well known in the art (e.g., PCR and othermolecular cloning methods). For example, PCR amplification usingsynthetic primers hybridizable to the 3′ and 5′ ends of a known sequencecan be performed using genomic DNA obtained from hybridoma cellsproducing the antibody of interest. Such PCR amplification methods canbe used to obtain nucleic acids comprising the sequence encoding thelight chain or heavy chain of an antibody. Such PCR amplificationmethods can be used to obtain nucleic acids comprising the sequenceencoding the variable light chain region or the variable heavy chainregion of an antibody. The amplified nucleic acids can be cloned intovectors for expression in host cells and for further cloning, forexample, to generate chimeric and humanized antibodies.

If a clone containing a nucleic acid encoding a particular antibody isnot available, but the sequence of the antibody molecule is known, anucleic acid encoding the immunoglobulin can be chemically synthesizedor obtained from a suitable source (e.g., an antibody cDNA library or acDNA library generated from, or nucleic acid, preferably poly A+RNA,isolated from, any tissue or cells expressing the antibody, such ashybridoma cells selected to express an antibody disclosed herein) by PCRamplification using synthetic primers hybridizable to the 3′ and 5′ endsof the sequence or by cloning using an oligonucleotide probe specificfor the particular gene sequence to identify, e.g., a cDNA clone from acDNA library that encodes the antibody. Amplified nucleic acidsgenerated by PCR can then be cloned into replicable cloning vectorsusing any method well known in the art.

DNA encoding anti-ApoC3 (e.g., human ApoC3) antibodies disclosed hereincan be readily isolated and sequenced using conventional procedures(e.g., by using oligonucleotide probes that are capable of bindingspecifically to genes encoding the heavy and light chains of theanti-ApoC3 (e.g., human ApoC3) antibodies). Hybridoma cells can serve asa source of such DNA. Once isolated, the DNA can be placed intoexpression vectors, which are then transfected into host cells such asE. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells(e.g., CHO cells from the CHO GS System™ (Lonza)), or myeloma cells thatdo not otherwise produce immunoglobulin protein, to obtain the synthesisof anti-ApoC3 (e.g., human ApoC3) antibodies in the recombinant hostcells.

To generate whole antibodies, PCR primers including VH or VL nucleotidesequences, a restriction site, and a flanking sequence to protect therestriction site can be used to amplify the VH or VL sequences in scFvclones. Utilizing cloning techniques known to those of skill in the art,the PCR amplified VH domains can be cloned into vectors expressing aheavy chain constant region, e.g., the human gamma 4 constant region,and the PCR amplified VL domains can be cloned into vectors expressing alight chain constant region, e.g., human kappa or lambda constantregions. In certain embodiments, the vectors for expressing the VH or VLdomains comprise an EF-1α promoter, a secretion signal, a cloning sitefor the variable region, constant domains, and a selection marker suchas neomycin. The VH and VL domains can also be cloned into one vectorexpressing the necessary constant regions. The heavy chain conversionvectors and light chain conversion vectors are then co-transfected intocell lines to generate stable or transient cell lines that expressfull-length antibodies, e.g., IgG, using techniques known to those ofskill in the art.

The DNA also can be modified, for example, by substituting the codingsequence for human heavy and light chain constant domains in place ofthe murine sequences, or by covalently joining to the immunoglobulincoding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide.

Also provided are polynucleotides that hybridize under high stringency,intermediate or lower stringency hybridization conditions topolynucleotides that encode an antibody disclosed herein. In specificembodiments, polynucleotides disclosed herein hybridize under highstringency, intermediate or lower stringency hybridization conditions topolynucleotides encoding a VH domain or VL domain provided herein.

Hybridization conditions have been described in the art and are known toone of skill in the art. For example, hybridization under stringentconditions can involve hybridization to filter-bound DNA in 6× sodiumchloride/sodium citrate (SSC) at about 45° C. followed by one or morewashes in 0.2×SSC/0.1% SDS at about 50-65° C.; hybridization underhighly stringent conditions can involve hybridization to filter-boundnucleic acid in 6×SSC at about 45° C. followed by one or more washes in0.1×SSC/0.2% SDS at about 68° C. Hybridization under other stringenthybridization conditions are known to those of skill in the art and havebeen described, see, for example, Ausubel F M et al., eds., (1989)Current Protocols in Molecular Biology, Vol. I, Green PublishingAssociates, Inc. and John Wiley & Sons, Inc., New York at pages6.3.1-6.3.6 and 2.10.3.

In certain aspects, provided herein are cells (e.g., host cells)expressing (e.g., recombinantly) antibodies disclosed herein whichspecifically bind to ApoC3 (e.g., human ApoC3) and relatedpolynucleotides and expression vectors. Provided herein are vectors(e.g., expression vectors) comprising polynucleotides comprisingnucleotide sequences encoding anti-ApoC3 (e.g., human ApoC3) antibodiesor a fragment for recombinant expression in host cells, preferably inmammalian cells. Also provided herein are host cells comprising suchvectors for recombinantly expressing anti-ApoC3 (e.g., human ApoC3)antibodies disclosed herein (e.g., human or humanized antibody). In aparticular aspect, provided herein are methods for producing an antibodydisclosed herein, comprising expressing such antibody from a host cell.

Recombinant expression of an antibody disclosed herein (e.g., afull-length antibody, heavy or light chain of an antibody, or a singlechain antibody disclosed herein) that specifically binds to ApoC3 (e.g.,human ApoC3) involves construction of an expression vector containing apolynucleotide that encodes the antibody. Once a polynucleotide encodingan antibody molecule, heavy or light chain of an antibody, (e.g., heavyor light chain variable regions) disclosed herein has been obtained, thevector for the production of the antibody molecule can be produced byrecombinant DNA technology using techniques well known in the art. Thus,methods for preparing a protein by expressing a polynucleotidecontaining an antibody or antibody fragment (e.g., light chain or heavychain) encoding nucleotide sequence are disclosed herein. Methods whichare well known to those skilled in the art can be used to constructexpression vectors containing antibody or antibody fragment (e.g., lightchain or heavy chain) coding sequences and appropriate transcriptionaland translational control signals.

These methods include, for example, in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. Also providedare replicable vectors comprising a nucleotide sequence encoding anantibody molecule disclosed herein, a heavy or light chain of anantibody, a heavy or light chain variable region of an antibody, or aheavy or light chain CDR, operably linked to a promoter. Such vectorscan, for example, include the nucleotide sequence encoding the constantregion of the antibody molecule (see, e.g., International PublicationNos. WO 86/05807 and WO 89/01036; and U.S. Pat. No. 5,122,464) andvariable regions of the antibody can be cloned into such a vector forexpression of the entire heavy, the entire light chain, or both theentire heavy and light chains.

An expression vector can be transferred to a cell (e.g., host cell) byconventional techniques and the resulting cells can then be cultured byconventional techniques to produce an antibody disclosed herein. Thus,provided herein are host cells containing a polynucleotide encoding anantibody disclosed herein, or a heavy or light chain thereof, orfragment thereof, or a single chain antibody disclosed herein, operablylinked to a promoter for expression of such sequences in the host cell.In certain embodiments, for the expression of double-chained antibodies,vectors encoding both the heavy and light chains, individually, can beco-expressed in the host cell for expression of the entireimmunoglobulin molecule, as detailed below. In certain embodiments, ahost cell contains a vector comprising a polynucleotide encoding boththe heavy chain and light chain of an antibody disclosed herein. Inspecific embodiments, a host cell contains two different vectors, afirst vector comprising a polynucleotide encoding a heavy chain or aheavy chain variable region of an antibody disclosed herein, or afragment thereof, and a second vector comprising a polynucleotideencoding a light chain or a light chain variable region of an antibodydisclosed herein, or a fragment thereof. In other embodiments, a firsthost cell comprises a first vector comprising a polynucleotide encodinga heavy chain or a heavy chain variable region of an antibody disclosedherein, or a fragment thereof, and a second host cell comprises a secondvector comprising a polynucleotide encoding a light chain or a lightchain variable region of an antibody disclosed herein. In specificembodiments, a heavy chain/heavy chain variable region expressed by afirst cell associated with a light chain/light chain variable region ofa second cell to form an anti-ApoC3 antibody disclosed herein. Incertain embodiments, provided herein is a population of host cellscomprising such first host cell and such second host cell.

In a particular embodiment, provided herein is a population of vectorscomprising a first vector comprising a polynucleotide encoding a lightchain/light chain variable region of an anti-ApoC3 antibody disclosedherein, and a second vector comprising a polynucleotide encoding a heavychain/heavy chain variable region of an anti-ApoC3 antibody disclosedherein.

A variety of host-expression vector systems can be utilized to expressantibody molecules disclosed herein (see, e.g., U.S. Pat. No.5,807,715). Such host-expression systems represent vehicles by which thecoding sequences of interest can be produced and subsequently purified,but also represent cells which can, when transformed or transfected withthe appropriate nucleotide coding sequences, express an antibodymolecule disclosed herein in situ. These include but are not limited tomicroorganisms such as bacteria (e.g., E. coli and B. subtilis)transformed with recombinant bacteriophage DNA, plasmid DNA or cosmidDNA expression vectors containing antibody coding sequences; yeast(e.g., Saccharomyces Pichia) transformed with recombinant yeastexpression vectors containing antibody coding sequences; insect cellsystems infected with recombinant virus expression vectors (e.g.,baculovirus) containing antibody coding sequences; plant cell systems(e.g., green algae such as Chlamydomonas reinhardtii) infected withrecombinant virus expression vectors (e.g., cauliflower mosaic virus,CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmidexpression vectors (e.g., Ti plasmid) containing antibody codingsequences; or mammalian cell systems (e.g., COS (e.g., COS1 or COS),CHO, BHK, MDCK, HEK 293, NS0, PER.C6, VERO, CRL7O3O, HsS78Bst, HeLa, andNIH 3T3, HEK-293T, HepG2, SP210, R1.1, B-W, L-M, BSC1, BSC40, YB/20 andBMT10 cells) harboring recombinant expression constructs containingpromoters derived from the genome of mammalian cells (e.g.,metallothionein promoter) or from mammalian viruses (e.g., theadenovirus late promoter; the vaccinia virus 7.5K promoter). In aspecific embodiment, cells for expressing antibodies disclosed hereinare CHO cells, for example CHO cells from the CHO GS System™ (Lonza). Ina particular embodiment, cells for expressing antibodies disclosedherein are human cells, e.g., human cell lines. In a specificembodiment, a mammalian expression vector is pOptiVEC™ or pcDNA3.3. In aparticular embodiment, bacterial cells such as Escherichia coli, oreukaryotic cells (e.g., mammalian cells), especially for the expressionof whole recombinant antibody molecule, are used for the expression of arecombinant antibody molecule. For example, mammalian cells such asChinese hamster ovary (CHO) cells, in conjunction with a vector such asthe major intermediate early gene promoter element from humancytomegalovirus is an effective expression system for antibodies(Foecking M K & Hofstetter H (1986) Gene 45: 101-5; and Cockett M I etal., (1990) Biotechnology 8(7): 662-7). In certain embodiments,antibodies disclosed herein are produced by CHO cells or NS0 cells. In aspecific embodiment, the expression of nucleotide sequences encodingantibodies disclosed herein which specifically bind ApoC3 (e.g., humanApoC3) is regulated by a constitutive promoter, inducible promoter ortissue specific promoter.

In bacterial systems, a number of expression vectors can beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such anantibody is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified can be desirable. Such vectors include, but are not limited to,the E. coli expression vector pUR278 (Ruether U & Mueller-Hill B (1983)EMBO J 2: 1791-1794), in which the antibody coding sequence can beligated individually into the vector in frame with the lac Z codingregion so that a fusion protein is produced; pIN vectors (Inouye S &Inouye M (1985) Nuc Acids Res 13: 3101-3109; Van Heeke G & Schuster S M(1989) J Biol Chem 24: 5503-5509); and the like. For example, pGEXvectors can also be used to express foreign polypeptides as fusionproteins with glutathione 5-transferase (GST). In general, such fusionproteins are soluble and can easily be purified from lysed cells byadsorption and binding to matrix glutathione agarose beads followed byelution in the presence of free glutathione. The pGEX vectors aredesigned to include thrombin or factor Xa protease cleavage sites sothat the cloned target gene product can be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV), for example, can be used as a vector to express foreign genes.The virus grows in Spodoptera frugiperda cells. The antibody codingsequence can be cloned individually into non-essential regions (forexample the polyhedrin gene) of the virus and placed under control of anAcNPV promoter (for example the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems canbe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest can be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene can then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing the antibody molecule in infected hosts (e.g., see Logan J &Shenk T (1984) PNAS 81(12): 3655-9). Specific initiation signals canalso be required for efficient translation of inserted antibody codingsequences. These signals include the ATG initiation codon and adjacentsequences. Furthermore, the initiation codon must be in phase with thereading frame of the desired coding sequence to ensure translation ofthe entire insert. These exogenous translational control signals andinitiation codons can be of a variety of origins, both natural andsynthetic. The efficiency of expression can be enhanced by the inclusionof appropriate transcription enhancer elements, transcriptionterminators, etc. (see, e.g., Bitter G et al., (1987) Methods Enzymol.153: 516-544).

In addition, a host cell strain can be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products canbe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product can be used. Such mammalian hostcells include but are not limited to CHO, VERO, BHK, Hela, MDCK, HEK293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NS0 (a murinemyeloma cell line that does not endogenously produce any immunoglobulinchains), CRL7O3O, COS (e.g., COS1 or COS), PER.C6, VERO, HsS78Bst,HEK-293T, HepG2, SP210, R1.1, B-W, L-M, BSC1, BSC40, YB/20, BMT10 andHsS78Bst cells. In certain embodiments, anti-ApoC3 (e.g., human ApoC3)antibodies disclosed herein are produced in mammalian cells, such as CHOcells.

In a specific embodiment, the antibodies disclosed herein have reducedfucose content or no fucose content. Such antibodies can be producedusing techniques known one skilled in the art. For example, theantibodies can be expressed in cells deficient or lacking the ability ofto fucosylate. In a specific example, cell lines with a knockout of bothalleles of α1,6-fucosyltransferase can be used to produce antibodieswith reduced fucose content. The Potelligent® system (Lonza) is anexample of such a system that can be used to produce antibodies withreduced fucose content.

For long-term, high-yield production of recombinant proteins, stableexpression cells can be generated. For example, cell lines which stablyexpress an anti-ApoC3 antibody disclosed herein can be engineered. Inspecific embodiments, a cell provided herein stably expresses a lightchain/light chain variable region and a heavy chain/heavy chain variableregion which associate to form an antibody disclosed herein.

In certain aspects, rather than using expression vectors which containviral origins of replication, host cells can be transformed with DNAcontrolled by appropriate expression control elements (e.g., promoter,enhancer, sequences, transcription terminators, polyadenylation sites,etc.), and a selectable marker. Following the introduction of theforeign DNA/polynucleotide, engineered cells can be allowed to grow for1-2 days in an enriched media, and then are switched to a selectivemedia. The selectable marker in the recombinant plasmid confersresistance to the selection and allows cells to stably integrate theplasmid into their chromosomes and grow to form foci which in turn canbe cloned and expanded into cell lines. This method can advantageouslybe used to engineer cell lines which express an anti-ApoC3 antibodydisclosed herein. Such engineered cell lines can be particularly usefulin screening and evaluation of compositions that interact directly orindirectly with the antibody molecule.

A number of selection systems can be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler M et al., (1977) Cell11(1): 223-32), hypoxanthineguanine phosphoribosyltransferase (SzybalskaE H & Szybalski W (1962) PNAS 48(12): 2026-2034) and adeninephosphoribosyltransferase (Lowy I et al., (1980) Cell 22(3): 817-23)genes in tk-, hgprt- or aprt-cells, respectively. Also, antimetaboliteresistance can be used as the basis of selection for the followinggenes: dhfr, which confers resistance to methotrexate (Wigler M et al.,(1980) PNAS 77(6): 3567-70; O'Hare K et al., (1981) PNAS 78: 1527-31);gpt, which confers resistance to mycophenolic acid (Mulligan R C & BergP (1981) PNAS 78(4): 2072-6); neo, which confers resistance to theaminoglycoside G-418 (Wu G Y & Wu C H (1991) Biotherapy 3: 87-95;Tolstoshev P (1993) Ann Rev Pharmacol Toxicol 32: 573-596; Mulligan R C(1993) Science 260: 926-932; and Morgan R A & Anderson W F (1993) AnnRev Biochem 62: 191-217; Nabel G J & Felgner P L (1993) TrendsBiotechnol 11(5): 211-5); and hygro, which confers resistance tohygromycin (Santerre R F et al., (1984) Gene 30(1-3): 147-56). Methodscommonly known in the art of recombinant DNA technology can be routinelyapplied to select the desired recombinant clone and such methods aredescribed, for example, in Ausubel F M et al., (eds.), Current Protocolsin Molecular Biology, John Wiley & Sons, N Y (1993); Kriegler M, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, N Y(1990); and in Chapters 12 and 13, Dracopoli N C et al., (eds.), CurrentProtocols in Human Genetics, John Wiley & Sons, N Y (1994);Colbére-Garapin F et al., (1981) J Mol Biol 150: 1-14, which areincorporated by reference herein in their entireties.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see Bebbington C R & Hentschel C C G, Theuse of vectors based on gene amplification for the expression of clonedgenes in mammalian cells in DNA cloning, Vol. 3 (Academic Press, NewYork, 1987)). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse G F et al., (1983) Mol Cell Biol3: 257-66).

The host cell can be co-transfected with two or more expression vectorsdescribed herein, the first vector encoding a heavy chain derivedpolypeptide and the second vector encoding a light chain derivedpolypeptide. The two vectors can contain identical selectable markerswhich enable equal expression of heavy and light chain polypeptides. Thehost cells can be co-transfected with different amounts of the two ormore expression vectors. For example, host cells can be transfected withany one of the following ratios of a first expression vector and asecond expression vector: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9,1:10, 1:12, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50.

Alternatively, a single vector can be used which encodes, and is capableof expressing, both heavy and light chain polypeptides. In suchsituations, the light chain should be placed before the heavy chain toavoid an excess of toxic free heavy chain (Proudfoot N J (1986) Nature322: 562-565; and Kohler G (1980) PNAS 77: 2197-2199). The codingsequences for the heavy and light chains can comprise cDNA or genomicDNA. The expression vector can be monocistronic or multicistronic. Amulticistronic nucleic acid construct can encode 2, 3, 4, 5, 6, 7, 8, 9,10 or more, or in the range of 2-5, 5-10 or 10-20 genes/nucleotidesequences. For example, a bicistronic nucleic acid construct cancomprise in the following order a promoter, a first gene (e.g., heavychain of an antibody disclosed herein), and a second gene and (e.g.,light chain of an antibody disclosed herein). In such an expressionvector, the transcription of both genes can be driven by the promoter,whereas the translation of the mRNA from the first gene can be by acap-dependent scanning mechanism and the translation of the mRNA fromthe second gene can be by a cap-independent mechanism, e.g., by an IRES.

Once an antibody molecule disclosed herein has been produced byrecombinant expression, it can be purified by any method known in theart for purification of an immunoglobulin molecule, for example, bychromatography (e.g., ion exchange, affinity, particularly by affinityfor the specific antigen after Protein A, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins. Further, theantibodies disclosed herein can be fused to heterologous polypeptidesequences disclosed herein or otherwise known in the art to facilitatepurification.

In specific embodiments, an antibody disclosed herein is isolated orpurified. Generally, an isolated antibody is one that is substantiallyfree of other antibodies with different antigenic specificities than theisolated antibody. For example, in a particular embodiment, apreparation of an antibody disclosed herein is substantially free ofcellular material or chemical precursors. The language “substantiallyfree of cellular material” includes preparations of an antibody in whichthe antibody is separated from cellular components of the cells fromwhich it is isolated or recombinantly produced. Thus, an antibody thatis substantially free of cellular material includes preparations ofantibody having less than about 30%, 20%, 10%, 5%, 2%, 1%, 0.5%, or 0.1%(by dry weight) of heterologous protein (also referred to herein as a“contaminating protein”) or variants of an antibody, for example,different post-translational modified forms of an antibody or otherdifferent versions of an antibody (e.g., antibody fragments). When theantibody is recombinantly produced, it is also generally substantiallyfree of culture medium, i.e., culture medium represents less than about20%, 10%, 2%, 1%, 0.5%, or 0.1% of the volume of the proteinpreparation. When the antibody is produced by chemical synthesis, it isgenerally substantially free of chemical precursors or other chemicals,i.e., it is separated from chemical precursors or other chemicals whichare involved in the synthesis of the protein. Accordingly, suchpreparations of the antibody have less than about 30%, 20%, 10%, or 5%(by dry weight) of chemical precursors or compounds other than theantibody of interest. In a specific embodiment, antibodies disclosedherein are isolated or purified.

Antibodies that specifically bind to ApoC3 (e.g., human ApoC3) can beproduced by any method known in the art for the synthesis of antibodies,for example, by chemical synthesis or by recombinant expressiontechniques. The methods disclosed herein employs, unless otherwiseindicated, conventional techniques in molecular biology, microbiology,genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR,oligonucleotide synthesis and modification, nucleic acid hybridization,and related fields within the skill of the art. These techniques aredescribed, for example, in the references cited herein and are fullyexplained in the literature. See, e.g., Maniatis T et al., (1982)Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress; Sambrook J et al., (1989), Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor Laboratory Press; Sambrook Jet al., (2001) Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.; Ausubel F M et al.,Current Protocols in Molecular Biology, John Wiley & Sons (1987 andannual updates); Current Protocols in Immunology, John Wiley & Sons(1987 and annual updates) Gait (ed.) (1984) Oligonucleotide Synthesis: APractical Approach, IRL Press; Eckstein (ed.) (1991) Oligonucleotidesand Analogues: A Practical Approach, IRL Press; Birren B et al., (eds.)(1999) Genome Analysis: A Laboratory Manual, Cold Spring HarborLaboratory Press.

In a specific embodiment, an antibody disclosed herein is an antibody(e.g., recombinant antibody) prepared, expressed, created or isolated byany means that involves creation, e.g., via synthesis, geneticengineering of DNA sequences. In certain embodiments, such antibodycomprises sequences (e.g., DNA sequences or amino acid sequences) thatdo not naturally exist within the antibody germline repertoire of ananimal or mammal (e.g., human) in vivo.

In one aspect, provided herein is a method of making an antibody whichspecifically binds to ApoC3 (e.g., human ApoC3) comprising culturing acell or host cell disclosed herein. In a certain aspect, provided hereinis a method of making an antibody which specifically binds to ApoC3(e.g., human ApoC3) comprising expressing (e.g., recombinantlyexpressing) the antibody using a cell or host cell disclosed herein(e.g., a cell or a host cell comprising polynucleotides encoding anantibody disclosed herein). In a particular embodiment, the cell is anisolated cell. In a particular embodiment, the exogenous polynucleotideshave been introduced into the cell. In a particular embodiment, themethod further comprises the step of purifying the antibody obtainedfrom the cell or host cell.

Methods for producing polyclonal antibodies are known in the art (see,for example, Chapter 11 in: Short Protocols in Molecular Biology, (2002)5th Ed., Ausubel F M et al., eds., John Wiley and Sons, New York).

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow E & Lane D,Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press,2nd ed. 1988); Hammerling G J et al., in: Monoclonal Antibodies andT-Cell Hybridomas 563 681 (Elsevier, N.Y., 1981). The term “monoclonalantibody” as used herein is not limited to antibodies produced throughhybridoma technology. For example, monoclonal antibodies can be producedrecombinantly from host cells exogenously expressing an antibodydisclosed herein, for example, light chain or heavy chain of suchantibody.

In specific embodiments, a “monoclonal antibody,” as used herein, is anantibody produced by a single cell (e.g., hybridoma or host cellproducing a recombinant antibody), wherein the antibody specificallybinds to ApoC3 (e.g., human ApoC3) as determined, e.g., by ELISA orother antigen-binding or competitive binding assay known in the art orin the examples provided herein. In particular embodiments, a monoclonalantibody can be a chimeric antibody or a humanized antibody. In certainembodiments, a monoclonal antibody is a monovalent antibody ormultivalent (e.g., bivalent) antibody. In particular embodiments, amonoclonal antibody is a monospecific or multispecific antibody (e.g.,bispecific antibody). Monoclonal antibodies disclosed herein can, forexample, be made by the hybridoma method as described in Kohler G &Milstein C (1975) Nature 256: 495 or can, e.g., be isolated from phagelibraries using the techniques as disclosed herein, for example. Othermethods for the preparation of clonal cell lines and of monoclonalantibodies expressed thereby are well known in the art (see, forexample, Chapter 11 in: Short Protocols in Molecular Biology, (2002) 5thEd., Ausubel F M et al., supra).

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art. For example,in the hybridoma method, a mouse or other appropriate host animal, suchas a sheep, goat, rabbit, rat, hamster or macaque monkey, is immunizedto elicit lymphocytes that produce or are capable of producingantibodies that will specifically bind to the protein (e.g., ApoC3(e.g., human ApoC3)) used for immunization. Alternatively, lymphocytesmay be immunized in vitro. Lymphocytes then are fused with myeloma cellsusing a suitable fusing agent, such as polyethylene glycol, to form ahybridoma cell (Goding J W (Ed), Monoclonal Antibodies: Principles andPractice, pp. 59-103 (Academic Press, 1986)). Additionally, a RIMMS(repetitive immunization multiple sites) technique can be used toimmunize an animal (Kilpatrick K E et al., (1997) Hybridoma 16:381-9,incorporated by reference in its entirety).

In some embodiments, mice (or other animals, such as rats, monkeys,donkeys, pigs, sheep, hamster, or dogs) can be immunized with an antigen(e.g., ApoC3 (e.g., human ApoC3)) and once an immune response isdetected, e.g., antibodies specific for the antigen are detected in themouse serum, the mouse spleen is harvested and splenocytes isolated. Thesplenocytes are then fused by well-known techniques to any suitablemyeloma cells, for example cells from cell line SP20 available from theAmerican Type Culture Collection (ATCC®) (Manassas, Va.), to formhybridomas. Hybridomas are selected and cloned by limited dilution. Incertain embodiments, lymph nodes of the immunized mice are harvested andfused with NS0 myeloma cells.

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Specific embodiments employ myeloma cells that fuse efficiently, supportstable high-level production of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. Among these myeloma cell lines are murine myeloma lines, such asNS0 cell line or those derived from MOPC-21 and MPC-11 mouse tumorsavailable from the Salk Institute Cell Distribution Center, San Diego,Calif., USA, and SP-2 or X63-Ag8.653 cells available from the AmericanType Culture Collection, Rockville, Md., USA. Human myeloma andmouse-human heteromyeloma cell lines also have been described for theproduction of human monoclonal antibodies (Kozbor D (1984) J Immunol133: 3001-5; Brodeur et al., Monoclonal Antibody Production Techniquesand Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against ApoC3 (e.g., humanApoC3). The binding specificity of monoclonal antibodies produced byhybridoma cells is determined by methods known in the art, for example,immunoprecipitation or by an in vitro binding assay, such asradioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, or activity, the clones may be subclonedby limiting dilution procedures and grown by standard methods (Goding JW (Ed), Monoclonal Antibodies: Principles and Practice, supra). Suitableculture media for this purpose include, for example, D-MEM or RPMI 1640medium. In addition, the hybridoma cells may be grown in vivo as ascitestumors in an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

Antibodies disclosed herein include antibody fragments which recognizespecific ApoC3 (e.g., human ApoC3) and can be generated by any techniqueknown to those of skill in the art. For example, Fab and F(ab′)2fragments disclosed herein can be produced by proteolytic cleavage ofimmunoglobulin molecules, using enzymes such as papain (to produce Fabfragments) or pepsin (to produce F(ab′)2 fragments). A Fab fragmentcorresponds to one of the two identical arms of an antibody molecule andcontains the complete light chain paired with the VH and CH1 domains ofthe heavy chain. A F(ab′)2 fragment contains the two antigen-bindingarms of an antibody molecule linked by disulfide bonds in the hingeregion.

Further, the antibodies disclosed herein can also be generated usingvarious phage display methods known in the art. In phage displaymethods, functional antibody domains are displayed on the surface ofphage particles which carry the polynucleotide sequences encoding them.In particular, DNA sequences encoding VH and VL domains are amplifiedfrom animal cDNA libraries (e.g., human or murine cDNA libraries ofaffected tissues). The DNA encoding the VH and VL domains are recombinedtogether with a scFv linker by PCR and cloned into a phagemid vector.The vector is electroporated in E. coli and the E. coli is infected withhelper phage. Phage used in these methods are typically filamentousphage including fd and M13, and the VH and VL domains are usuallyrecombinantly fused to either the phage gene III or gene VIII. Phageexpressing an antigen binding domain that binds to a particular antigencan be selected or identified with antigen, e.g., using labeled antigenor antigen bound or captured to a solid surface or bead. Examples ofphage display methods that can be used to make the antibodies disclosedherein include those disclosed in Brinkman U et al., (1995) J ImmunolMethods 182: 41-50; Ames R S et al., (1995) J Immunol Methods 184:177-186; Kettleborough C A et al., (1994) Eur J Immunol 24: 952-958;Persic L et al., (1997) Gene 187: 9-18; Burton D R & Barbas C F (1994)Advan Immunol 57: 191-280; PCT Application No. PCT/GB91/001134;International Publication Nos. WO 90/02809, WO 91/10737, WO 92/01047, WO92/18619, WO 93/1 1236, WO 95/15982, WO 95/20401, and WO 97/13844; andU.S. Pat. Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908,5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225,5,658,727, 5,733,743 and 5,969,108.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described below. Techniques to recombinantly produceantibody fragments such as Fab, Fab′ and F(ab′)2 fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication No. WO 92/22324; Mullinax R L et al., (1992) BioTechniques12(6): 864-9; Sawai H et al., (1995) Am J Reprod Immunol 34: 26-34; andBetter M et al., (1988) Science 240: 1041-1043.

In certain embodiments, to generate whole antibodies, PCR primersincluding VH or VL nucleotide sequences, a restriction site, and aflanking sequence to protect the restriction site can be used to amplifythe VH or VL sequences from a template, e.g., scFv clones. Utilizingcloning techniques known to those of skill in the art, the PCR amplifiedVH domains can be cloned into vectors expressing a VH constant region,and the PCR amplified VL domains can be cloned into vectors expressing aVL constant region, e.g., human kappa or lambda constant regions. The VHand VL domains can also be cloned into one vector expressing thenecessary constant regions. The heavy chain conversion vectors and lightchain conversion vectors are then co-transfected into cell lines togenerate stable or transient cell lines that express full-lengthantibodies, e.g., IgG, using techniques known to those of skill in theart.

A chimeric antibody is a molecule in which different portions of theantibody are derived from different immunoglobulin molecules. Forexample, a chimeric antibody can contain a variable region of a mouse orrat monoclonal antibody fused to a constant region of a human antibody.Methods for producing chimeric antibodies are known in the art. See,e.g., Morrison S L (1985) Science 229: 1202-7; Oi V T & Morrison S L(1986) BioTechniques 4: 214-221; Gillies S D et al., (1989) J ImmunolMethods 125: 191-202; and U.S. Pat. Nos. 5,807,715, 4,816,567,4,816,397, and 6,331,415.

A humanized antibody is capable of binding to a predetermined antigenand which comprises a framework region having substantially the aminoacid sequence of a human immunoglobulin and CDRs having substantiallythe amino acid sequence of a non-human immunoglobulin (e.g., a murineimmunoglobulin). In particular embodiments, a humanized antibody alsocomprises at least a portion of an immunoglobulin constant region (Fc),typically that of a human immunoglobulin. The antibody also can includethe CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. Ahumanized antibody can be selected from any class of immunoglobulins,including IgM, IgG, IgD, IgA and IgE, and any isotype, including IgG₁,IgG₂, IgG₃ and IgG₄. Humanized antibodies can be produced using avariety of techniques known in the art, including but not limited to,CDR-grafting (European Patent No. EP 239400; International PublicationNo. WO 91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and5,585,089), veneering or resurfacing (European Patent Nos. EP 592106 andEP 519596; Padlan E A (1991) Mol Immunol 28(4/5): 489-498; Studnicka G Met al., (1994) Prot Engineering 7(6): 805-814; and Roguska M A et al.,(1994) PNAS 91: 969-973), chain shuffling (U.S. Pat. No. 5,565,332), andtechniques disclosed in, e.g., U.S. Pat. Nos. 6,407,213, 5,766,886,International Publication No. WO 93/17105; Tan P et al., (2002) JImmunol 169: 1119-25; Caldas C et al., (2000) Protein Eng. 13(5):353-60; Morea V et al., (2000) Methods 20(3): 267-79; Baca M et al.,(1997) J Biol Chem 272(16): 10678-84; Roguska M A et al., (1996) ProteinEng 9(10): 895 904; Couto J R et al., (1995) Cancer Res. 55 (23 Supp):5973s-5977s; Couto J R et al., (1995) Cancer Res 55(8): 1717-22; SandhuJ S (1994) Gene 150(2): 409-10 and Pedersen J T et al., (1994) J MolBiol 235(3): 959-73. See also U.S. Application Publication No. US2005/0042664 A1 (Feb. 24, 2005), which is incorporated by referenceherein in its entirety.

Methods for making multispecific (e.g., bispecific antibodies) have beendescribed, see, for example, U.S. Pat. Nos. 7,951,917; 7,183,076;8,227,577; 5,837,242; 5,989,830; 5,869,620; 6,132,992 and 8,586,713.

Single domain antibodies, for example, antibodies lacking the lightchains, can be produced by methods well known in the art. See RiechmannL & Muyldermans S (1999) J Immunol 231: 25-38; Nuttall S D et al.,(2000) Curr Pharm Biotechnol 1(3): 253-263; Muyldermans S, (2001) JBiotechnol 74(4): 277-302; U.S. Pat. No. 6,005,079; and InternationalPublication Nos. WO 94/04678, WO 94/25591 and WO 01/44301.

Further, antibodies that specifically bind to ApoC3 (e.g., human ApoC3)can, in turn, be utilized to generate anti-idiotype antibodies that“mimic” an antigen using techniques well known to those skilled in theart. (See, e.g., Greenspan N S & Bona C A (1989) FASEB J 7(5): 437-444;and Nissinoff A (1991) J Immunol 147(8): 2429-2438).

In particular embodiments, an antibody disclosed herein, which binds tothe same epitope of ApoC3 (e.g., human ApoC3) as an anti-ApoC3 antibodydisclosed herein, is a human antibody. In particular embodiments, anantibody disclosed herein, which competitively blocks (e.g., in adose-dependent manner) any one of the antibodies disclosed herein, frombinding to ApoC3 (e.g., human ApoC3), is a human antibody. Humanantibodies can be produced using any method known in the art. Forexample, transgenic mice which are incapable of expressing functionalendogenous immunoglobulins, but which can express human immunoglobulingenes, can be used. In particular, the human heavy and light chainimmunoglobulin gene complexes can be introduced randomly or byhomologous recombination into mouse embryonic stem cells. Alternatively,the human variable region, constant region, and diversity region can beintroduced into mouse embryonic stem cells in addition to the humanheavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes can be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of the Jxregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then bred to produce homozygousoffspring which express human antibodies. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of an antigen (e.g., ApoC3 (e.g., human ApoC3)). Monoclonalantibodies directed against the antigen can be obtained from theimmunized, transgenic mice using conventional hybridoma technology. Thehuman immunoglobulin transgenes harbored by the transgenic micerearrange during B cell differentiation, and subsequently undergo classswitching and somatic mutation. Thus, using such a technique, it ispossible to produce therapeutically useful IgG, IgA, IgM and IgEantibodies. For an overview of this technology for producing humanantibodies, see Lonberg N & Huszar D (1995) Int Rev Immunol 13:65-93.For a detailed discussion of this technology for producing humanantibodies and human monoclonal antibodies and protocols for producingsuch antibodies, see, e.g., International Publication Nos. WO 98/24893,WO 96/34096 and WO 96/33735; and U.S. Pat. Nos. 5,413,923, 5,625,126,5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318 and 5,939,598.Examples of mice capable of producing human antibodies include theXenomouse™ (Abgenix, Inc.; U.S. Pat. Nos. 6,075,181 and 6,150,184), theHuAb-Mouse™ (Mederex, Inc./Gen Pharm; U.S. Pat. Nos. 5,545,806 and5,569,825), the Trans Chromo Mouse™ (Kirin) and the KM Mouse™(Medarex/Kirin).

Human antibodies which specifically bind to ApoC3 (e.g., human ApoC3)can be made by a variety of methods known in the art including phagedisplay methods described above using antibody libraries derived fromhuman immunoglobulin sequences. See also U.S. Pat. Nos. 4,444,887,4,716,111, and 5,885,793; and International Publication Nos. WO98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO96/33735, and WO 91/10741.

In some embodiments, human antibodies can be produced using mouse-humanhybridomas. For example, human peripheral blood lymphocytes transformedwith Epstein-Barr virus (EBV) can be fused with mouse myeloma cells toproduce mouse-human hybridomas secreting human monoclonal antibodies,and these mouse-human hybridomas can be screened to determine ones whichsecrete human monoclonal antibodies that specifically bind to a targetantigen (e.g., ApoC3 (e.g., human ApoC3)). Such methods are known andare described in the art, see, e.g., Shinmoto H et al., (2004)Cytotechnology 46: 19-23; Naganawa Y et al., (2005) Human Antibodies 14:27-31.

6. Kits

Also provided, are kits comprising one or more antibodies disclosedherein, or pharmaceutical composition or conjugates thereof. In aspecific embodiment, provided herein is a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions disclosed herein, such asone or more antibodies provided herein. In some embodiments, the kitscontain a pharmaceutical composition disclosed herein and anyprophylactic or therapeutic agent, such as those disclosed herein.Optionally associated with such container(s) can be a notice in the formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of manufacture, use or sale for humanadministration.

Also provided, are kits that can be used in the above methods. In oneembodiment, a kit comprises an antibody disclosed herein, preferably apurified antibody, in one or more containers. In a specific embodiment,kits disclosed herein contain a substantially isolated ApoC3 (e.g.,human ApoC3) antigen as a control. In another specific embodiment, thekits disclosed herein further comprise a control antibody which does notreact with an ApoC3 (e.g., human ApoC3) antigen. In another specificembodiment, kits disclosed herein contain one or more elements fordetecting the binding of an antibody to ApoC3 (e.g., human ApoC3)antigen (e.g., the antibody can be conjugated to a detectable substratesuch as a fluorescent compound, an enzymatic substrate, a radioactivecompound or a luminescent compound, or a second antibody whichrecognizes the first antibody can be conjugated to a detectablesubstrate). In specific embodiments, a kit provided herein can include arecombinantly produced or chemically synthesized ApoC3 (e.g., humanApoC3) antigen. The ApoC3 (e.g., human ApoC3) antigen provided in thekit can also be attached to a solid support. In a more specificembodiment, the detecting means of the above described kit includes asolid support to which an ApoC3 (e.g., human ApoC3) antigen is attached.Such a kit can also include a non-attached reporter-labeled anti-humanantibody or anti-mouse/rat antibody. In this embodiment, binding of theantibody to the ApoC3 (e.g., human ApoC3) antigen can be detected bybinding of the said reporter-labeled antibody.

EXAMPLES

The previously identified antibody clone, 5E5, binds to ApoC3 with highaffinity at pH 7.4 and slightly reduced affinity at pH 5.5 (see U.S.provisional application 62/360,084). The instant disclosure providesnovel derivatives of clone 5E5 that exhibit high affinity binding toApoC3 at pH 7.4, but much reduced affinity to ApoC3 at pH 5.5 relativeto 5E5. The following examples describe the characterization of thenovel 5E5 derivatives. The amino acid sequences of 5E5 are set forth inU.S. provisional application 62/360,084, and the amino sequences of thenovel 5E5 derivatives are set forth in Tables 1-7, herein.

The examples in this Section are provided to further elucidate theadvantages and features of the present application, but are not intendedto limit the scope of the application. The examples are for illustrativepurposes only.

Example 1: In Vitro Characterization of Anti-ApoC3 scFv-Fc Antibodies

This example describes surface plasmon resonance (SPR)-based experimentsto determine the antigen-binding kinetics, at both pH 7.4 and pH 5.5, ofanti-ApoC3 scFv-Fc antibodies.

A panel of novel derivatives of antibody clone 5E5 was generated bysubstitution of one or more CDR amino acids in the VH and/or VL of 5E5with histidine. The antigen-binding kinetics, at both pH 7.4 and pH 5.5,of each 5E5 derivative was assessed using the SPR-based method set forthbelow, and clones exhibiting high affinity binding to ApoC3 at pH 7.4,but much reduced affinity to ApoC3 at pH 5.5 relative to 5E5 wereselected for further characterization. The binding kinetics of exemplary5E5 derivatives 5E5VH5_VLWT, 5E5VH12_VLWT, and 5E5VHWT_VL8 are set forthin Table 8.

Test antibodies were produced from transfected HEK293 cells at 50 mlsmall-scale cultures and purified by protein A chromatography using theÄKTA pure chromatography system. The quality and yields of the purifiedof the antibody fragments were determined by spectrophotometry and bySDS-PAGE.

An SPR-based method was employed, in which biotinylated human ApoC3 wascaptured on a streptavidin (SA) coated chip, and the binding kinetics oftest antibodies to the coated chip were measured at both pH 7.4 and pH5.5. Briefly, 20 μl of biotinylated human ApoC3 was injected at aconcentration of 10 μg/ml to reach a surface density of approximately500 RU. 60 μl of each test antibody was diluted in HBS-EP buffer (GE,cat. nr. BR-1008-26; 0.010 M HEPES, 0.150M NaCl, 3 mM EDTA, 0.05% (v/v)surfactant P20, pH 7.4), and was injected at a concentration of 1-100nM. The test antibodies were passed through the flow cells at a flowrate of 30 μl/min, followed by an off-rate wash at pH 7.4 or pH 5.5 for5 min. The resulting sensorgrams were analyzed using the BIAevaluation4.1 software applying the Langmuir 1:1 binding model to derive bindingkinetics. Data was zero adjusted and the reference cell sensorgrams weresubtracted.

TABLE 8 Binding kinetics of anti-ApoC3 scFv-Fc antibodies at pH 7.4 andpH 5.5 Association Rate Dissociation Rate Affinity ka (1/Ms) ka (1/Ms)kd (1/s) kd (1/s) K_(D) (nM) K_(D) (nM) K_(D) pH 5.5/ Antibody pH 7.4 pH5.5 pH 7.4 pH 5.5 pH 7.4 pH 5.5 K_(D) pH 7.4 5E5WT 8.23E+05 3.93E+051.75E−05 2.53E−05 0.02 0.06 3.0 5E5VH5_VLWT 1.78E+05 1.47E+05 1.05E−041.60E−03 0.59 10.90 18.5 5E5VH12_VLWT 3.08E+05 1.29E+05 1.57E−042.06E−03 0.51 16.00 31.4 5E5VHWT_VL8 3.81E+05 2.69E+05 2.21E−04 1.16E−030.58 4.29 7.4

All scFv-Fc antibodies tested exhibited higher affinity for ApoC3 at pH7.4 than at pH 5.5, with antibody 5E5VH12_VLWT showing the mostpronounced pH-dependent binding (see Table 8). The magnitude ofpH-dependent binding positively correlated with the dissociation rateunder acidic conditions.

Example 2: In Vitro Characterization of Anti-ApoC3 Human IgG₁ Antibodies

Based on the results in Example 1, test scFv-Fc antibodies weregenerated as human IgG₁ antibodies. An SPR-based assay was employed, inwhich human ApoC3 protein was immobilized on a CMS chip, and the bindingkinetics of test antibodies to the coated chip were measured at both pH7.4 and pH 5.5. Briefly, a solution of 50 μg/ml of native human ApoC3 in10 mM acetate buffer at pH 4.5 was prepared and injected until thesurface density reached approximately 500 RU. 60 μl of each testantibody was diluted in HBS-EP buffer (GE, cat. nr. BR-1008-26; 0.010 MHEPES, 0.150M NaCl, 3 mM EDTA, 0.05% (v/v) surfactant P20, pH 7.4), andwas injected at a concentration as described in Table 9. The testantibodies were passed through the flow cells at a flow rate of 30μl/min, followed by an off-rate wash at pH 7.4 or pH 5.5 for 5 min. Theresulting sensorgrams were analyzed using the BIAevaluation 4.1 softwareapplying the Langmuir 1:1 binding model to derive binding kinetics. Datawas zero adjusted and the reference cell sensorgrams were subtracted.

TABLE 9 Binding kinetics of anti ApoC3 human IgG₁ antibodies at pH 7.4and pH 5.5 Antibody pH ka (1/Ms) kd (1/s) Rmax (RU) Concentration K_(D)(M) 5E5WT 7.4 3.50E+05 1.79E−05 475 50-0.8 nM 5.12E−11 5.5 3.40E+051.70E−05 519 50-0.8 nM 4.99E−11 5E5VH5_VLWT 7.4 1.78E+05 3.77E−05 34750-0.8 nM 2.11E−10 5.5 1.55E+05 2.03E−04 313 50-0.8 nM 1.31E−095E5VH12_VLWT 7.4 3.93E+05 1.38E−05 420 50-0.8 nM 3.51E−11 5.5 3.66E+053.74E−04 413 50-0.8 nM 1.02E−09 5E5VHWT_VL8 7.4 7.82E+05 2.84E−05 47925-0.8 nM 3.63E−11 5.5 7.31E+05 4.06E−05 463 25-0.8 nM 5.56E−115E5VH5_VL8 7.4 1.77E+05 1.73E−04 309 50-0.8 nM 9.75E−10 5.5 2.11E+052.42E−03 183 50-0.8 nM 1.14E−08 5E5VH12_VL8 7.4 2.67E+05 1.63E−04 29150-0.8 nM 6.09E−10 5.5 1.88E+05 1.65E−03 523 50-0.8 nM 8.79E−095E5VH5VH12_VLWT 7.4 6.16E+04 3.05E−04 220 50-0.8 nM 4.96E−09 5.51.56E+05 3.16E−03 381 50-0.8 nM 2.02E−08 5E5VH5VH12_VL8 7.4 1.25E+052.80E−03 702 50-0.8 nM 2.24E+08 5.5 No binding detected

All antibodies tested bound to human ApoC3 at pH 7.4, and had reducedaffinity to ApoC3 at pH 5.5 (see Table 9). 5E5VH5_VLWT, 5E5VH12_VLWT,5E5VH5_VL8, and 5E5VH12 VL8 show particularly pronounced pH-dependence.

Example 3: Effect of Anti-ApoC3 Antibodies on VLDL Uptake by Hepatocytes

In this example, the ability of anti-ApoC3 antibodies to attenuate VLDLuptake by hepatocytes was determined.

Briefly, HepG2 cells (ATCC HB-8065) were cultured on a poly-d-lysinecoated surface in complete Minimum Essential Medium (MEM) supplementedwith 10% FCS for 24 hours, and in complete MEM supplemented with 0.0125%bovine serum albumin (MEM-BSA medium) for another 24 hours. The cellswere pre-incubated with 3 μM human ApoC3 protein (Athens Research andTechnology) and 3 μM of a test antibody in the IgG₁ format for 15minutes in fresh MEM-BSA medium, and 30 μg/mL ApoC3-depleted DiI-labeledVLDL (Kalen Biomedical, LLC #770130-9) was added to the medium. After a4-hour incubation, the cells were further incubated with fresh completeMEM supplemented with 1% intralipid for 20 minutes. The amount ofDiI-labeled VLDL taken up by the cells was determined by lysing thecells with isopropanol at room temperature for 15 minutes, measuringfluorescence of the DiI label in the lysate (ex=520 nm; em=580),calculating the amount of DiI-labeled VLDL using a standard curve, andnormalizing the data based on the quantity of total protein in thelysate. Data was graphed using GraphPad Prism 6 and is reported asaverage +/−SEM. One-way ANOVA with multiple comparisons were calculatedusing GraphPad Prism 6.

As shown in FIGS. 1A, 1B, and 1C, the 5E5WT, 5E5VHWT_VL8, 5E5VH5_VLWT,5E5VH12_VLWT, and 5E5VH5_VL8 antibodies increased VLDL uptake by HepG2cells. In particular, the 5E5VHWT_VL8, 5E5VH5_VLWT, 5E5VH12_VLWT, and5E5VH5_VL8 antibodies all completely restored VLDL uptake in thepresence of ApoC3.

Example 4: Pharmacokinetics and Pharmacodynamics of Anti-ApoC3Antibodies

This example describes the in vivo characterization of the 5E5VH5_VL8antibody using a mouse model having impaired triglyceride clearance dueto transgenic expression of human ApoC3.

4.1 Generation of Mouse Model

Wild-type C57BL/6 male mice aged 60-63 days maintained on a standardchow diet were infected with 3×10¹¹ viral particles of an AAV8 vectorharboring a human ApoC3 gene operably linked to a thyroxine bindingglobulin (TBG) promoter (RegenXBio) by intraperitoneal administration.Twelve days following the administration, blood samples were collectedfrom retro-orbital sinus, and levels of human ApoC3 in the blood sampleswere measured by ELISA using a primary anti-ApoC3 antibody (Abcam rabbitpolyclonal anti-human ApoC3 #ab21032) and a secondary ApoC3 antibody(Abcam goat polyclonal biotin-conjugate ApoC3 #ab21024). In the infectedmice, the mean serum level of human ApoC3 was 9.9 μM. The meancirculating triglyceride level after a four-hour fasting was 163 mg/dLin these mice, whereas the mean circulating triglyceride level incontrol mice was 109 mg/dL (p=0.0065).

The mice were then grouped such that all groups had similar mean ApoC3levels on Day 12. Fourteen days after the AAV infection, blood sampleswere collected from retro-orbital sinus to establish baseline (T=0)ApoC3 levels. 25 mg/kg of a test anti-ApoC3 human IgG₁ antibody wasadministered to each mouse by injection into the dorsal subcutaneousspace. An anti-hen egg lysosome human IgG₁ antibody (HyHELS) was used asan isotype control. Blood samples were collected from retro-orbitalsinus 0, 2, 4, 8, and 24 hours after the administration of the testantibodies, and approximately every 2 days afterwards for 30 days. Allanimal studies were carried out in accordance with the recommendationsin the Guide for the Care and Use of Laboratory Animals of the NationalInstitutes of Health. All procedures were approved by the InstitutionalAnimal Care and Use Committee of Vascumab, LLC.

4.2 Pharmacokinetics of Anti-ApoC3 Antibodies

Mice expressing human ApoC3 were generated and treated as described inSection 4.1. Plasma levels of the human IgG₁ antibodies were determinedwith an ELISA assay. Specifically, a 96-well plate (Griener #655061) wascoated overnight at 4° C. with 50 μL primary IgG antibody (Fitzgerald41-XG57 goat anti-human IgG Fc polyclonal) diluted in PBS. The plate waswashed 4 times with 200 μL TBS-T, and blocked with 200 μl of blockingbuffer consisting of 3% BSA (Roche BSA fraction V protease free #03 117332 001) plus clear milk (Pierce Clear Milk Blocker #37587) in PBS for90 minutes at 30° C. The blocking buffer was removed, and 50 μL of testsample diluted in blocking buffer was added and allowed to incubate for2 hours at room temperature with rotation at 300 rpm. The plate waswashed four times with 200 μL TBS-T, and 50 μL secondary antibody (Abcamgoat anti-human IgG-Fc (biotin) polyclonal #ab97223) diluted in blockingbuffer was added and allowed to incubate for 1 hour at room temperaturewith rotation at 300 rpm. The plate was washed once with TBS-T, and 50μL SA-HRP (Abcam #64269) diluted 100-fold in PBS was added and allowedto incubate for 30 min at RT with rotation at 300 rpm. The plate wasthen washed 4 times with 200 μL TBS-T, and developed with 80 μL TMB. Thechromogenic reaction was terminated by 50 μL 0.5 N HCL. Absorbance wasread at the wavelength of 450 nm. The amounts of human IgG in test wellswere calculated from a 4-parameter fit of a standard curve (MolecularDevices) constructed using the purified test antibody. This methoddetects human ApoC3 specifically, and does not cross-react with mouseApoC3.

As shown in FIG. 2A, the 5E5 antibody was rapidly degraded in miceexpressing human ApoC3. This can be explained by the rapid turnover ofApoC3 through uptake of ApoC3-containing lipid particles. The 5E5VH5_VL8antibody, which has a reduced affinity to ApoC3 at lower pH, candissociate from ApoC3 in acidic organelles and return to the bloodstreamvia endosomal recycling. The half-life of 5E5VH5_VL8 is about one week,which is similar to the half-life of the isotype control antibody HyHe15(an antibody that does not bind to a specific antigen in mice). Theplasma level of 5E5VH5_VL8 returned to the baseline about one monthafter the injection. The extended half-life of 5E5VH5_VL8 makes thisantibody an excellent candidate for clinical application, due to the lowfrequency of administration required to maintain a therapeutic level ofthe antibody in serum.

4.3 Pharmacodynamics of Anti-ApoC3 Antibodies

Mice expressing human ApoC3 were generated and treated as described inSection 4.1. Plasma levels of human ApoC3 and ApoB were determined withan ELISA assay. Specifically, a 96-well plate (Griener #655061) wascoated overnight at 4° C. with 50 μL primary ApoC3 antibody (Abcamrabbit polyclonal anti-human ApoC3 #ab21032) or 50 μL primary ApoBantibody (Meridian Life Sciences goat polyclonal anti-human ApoB#K45253G) diluted in PBS. The plate was washed 4 times with 200 μLTBS-T, and blocked with 200 μl of blocking buffer (Pierce Clear MilkBlocker #37587 in PBS) for 90 minutes at 30° C. The blocking buffer wasremoved, and 50 μL of test sample diluted in blocking buffer was addedand allowed to incubate for 2 hours at room temperature with rotation at300 rpm. The plate was washed four times with 200 μL TBS-T, and 50 μLsecondary ApoC3 antibody (Abcam goat polyclonal biotin-conjugate ApoC3#ab21024) or secondary ApoB antibody (Meridian Life Sciences goatpolyclonal biotin-conjugate ApoB48/100 #34003G) diluted in blockingbuffer was added and allowed to incubate for 1 hour at room temperaturewith rotation at 300 rpm. The plate was washed once with TBS-T, and 50μL SA-HRP (Abcam #64269) diluted 100-fold in PBS was added and allowedto incubate for 30 minutes at room temperature with rotation at 300 rpm.The plate was then washed 4 times with 200 μl TBS-T, and developed with80 μl TMB (Thermo Ultra-TMB ELISA #34028) followed by 50 μL 0.5 N HCL.Absorbance was read at 450 nm. The amount of ApoC3 in test wells wascalculated from a 4-parameter fit of a standard curve (MolecularDevices) constructed using purified ApoC3 (Athens Research andTechnology). The amount of ApoB in test wells was calculated from a4-parameter fit of a standard curve (Molecular Devices) constructedusing mouse VLDL isolated by centrifugation (ApoB content is assumed tobe 20% of total protein content). Data was calculated and plotted aspercentage values relative to the corresponding levels in mice treatedwith the HyHe15 control antibody.

As shown in FIGS. 2B and 2C, the 5E5 antibody reduced the plasma levelsof human ApoC3 and ApoB initially, but the levels returned to normalafter about 2 days. By contrast, 5E5VH5_VL8 reduced the plasma levels ofhuman ApoC3 and ApoB for about one month. This long duration of efficacywas consistent with the long half-life of 5E5VH5_VL8, and confirmed that5E5VH5_VL8 would be an excellent clinical candidate.

Example 5: Reduction of Fasting Triglyceride Levels and CirculatingPost-Prandial Triglyceride Levels by Anti-ApoC3 Antibodies

This example describes the reduction of fasting triglyceride levels andcirculating post-prandial triglyceride levels by the 5E5VH5_VL8 antibodyusing a mouse model having impaired triglyceride clearance due totransgenic expression of human ApoC3.

Wild-type C57BL/6 male mice aged 60-63 days maintained on a standardchow diet were infected with 3×10¹¹ viral particles of an AAV8 vectorharboring a human ApoC3 gene operably linked to a thyroxine bindingglobulin (TBG) promoter (RegenXBio) by intraperitoneal administration.Fourteen days following the administration, blood samples were collectedfrom retro-orbital sinus, and levels of human ApoC3 in the blood sampleswere measured by ELISA using a primary anti-ApoC3 antibody (Abcam rabbitpolyclonal anti-human ApoC3 #ab21032) and a secondary ApoC3 antibody(Abcam goat polyclonal biotin-conjugate ApoC3 #ab21024). The mice werethen grouped such that all groups had similar mean ApoC3 levels on Day14.

On Day 17, blood samples were collected from retro-orbital sinus, andthe mice were injected subcutaneously with 30 mg/kg of 5E5VH5_VL8 or theHyHe15 control antibody in the fed state (t=−24 hours). After providingthe Chow diet for another 6 hours, the mice were fasted for 18 hours,and blood samples at t=0 were collected from retro-orbital sinus. Themice were then challenged with a 10 mL/kg oral bolus of olive oil.Retro-orbital sinus blood samples were obtained at 1, 2, 3 and 4 hoursfollowing olive oil challenge.

The levels of plasma triglycerides were determined by a colorimetricassay using the Thermo Scientific™ Triglycerides Reagent (TR22421).Briefly, 10 μl of diluted plasma samples were incubated with 180 μLTriglycerides Reagent in a clear 96 well plate (Corning Costar 9017) at37° C. for 10 minutes. Absorbance at 540 nm was read on a Spectramax M2(Molecular Devices), and the triglyceride concentrations were calculatedfrom a linear fit (Softmax Pro, Molecular Devices) of a glycerolstandard curve. The area under the curve (AUC) values for plasmatriglyceride were calculated using GraphPad Prism 6. The levels of ApoC3were determined by the method described in section 4.2 above.

As shown in FIG. 3A, the fasting triglyceride levels in the mice treatedwith 5E5VH5_VL8 for 24 hours were significantly lower (by 22%, p=0.004)than the levels in the mice treated with the HyHe15 control antibody.After the olive oil challenge, the mice treated with 5E5VH5_VL8 showedlower increase in the plasma triglyceride levels (FIG. 3B), and the meanAUC value was reduced by 47% (p=0.04) compared to that of the micetreated with the HyHe15 control antibody (FIG. 3C). The circulatinglevels of human ApoC3 were also significantly reduced in the micetreated with 5E5VH5_VL8 relative to those of the mice treated with theHyHe15 control antibody (p values<0.001 at t=0, 1, 2, 3 and 4 hours postolive oil dose) (FIG. 3D).

The invention is not to be limited in scope by the specific embodimentsdisclosed herein. Indeed, various modifications of the invention inaddition to those described will become apparent to those skilled in theart from the foregoing description and accompanying figures. Suchmodifications are intended to fall within the scope of the appendedclaims.

All references (e.g., publications or patents or patent applications)cited herein are incorporated herein by reference in their entirety andfor all purposes to the same extent as if each individual reference(e.g., publication or patent or patent application) was specifically andindividually indicated to be incorporated by reference in its entiretyfor all purposes.

Other embodiments are within the following claims.

1.-39. (canceled)
 40. A polynucleotide encoding a heavy chain variableregion and/or a light chain variable region of an antibody, the antibodycomprising a heavy chain variable region comprising complementaritydetermining regions CDRH1, CDRH2 and CDRH3, and a light chain variableregion comprising complementarity determining regions CDRL1, CDRL2 andCDRL3, wherein: (a) CDRH1 comprises the amino acid sequence of SEQ IDNO: 3; (b) CDRH2 comprises the amino acid sequence ofSIX₁TDGGGTAYRDSVKG, wherein X₁ is S or H (SEQ ID NO: 4); (c) CDRH3comprises the amino acid sequence of X₂GYSD, wherein X₂ is A or H (SEQID NO: 5); (d) CDRL1 comprises the amino acid sequence of SEQ ID NO: 6;(e) CDRL2 comprises the amino acid sequence of SEQ ID NO: 7; and (f)CDRL3 comprises the amino acid sequence of AX₃GTYYPHT, wherein X₃ is Qor H (SEQ ID NO: 8), and wherein at least one of X₁, X₂, and X₃ is H.41. An expression vector comprising the polynucleotide of claim
 40. 42.A host cell comprising the expression vector of claim
 41. 43. A methodfor producing an antibody that specifically binds to ApoC3, the antibodycomprising a heavy chain variable region comprising complementaritydetermining regions CDRH1, CDRH2 and CDRH3, and a light chain variableregion comprising complementarity determining regions CDRL1, CDRL2 andCDRL3, wherein: (i) CDRH1 comprises the amino acid sequence of SEQ IDNO: 3; (ii) CDRH2 comprises the amino acid sequence ofSIX₁TDGGGTAYRDSVKG, wherein X₁ is S or H (SEQ ID NO: 4); (iii) CDRH3comprises the amino acid sequence of X₂GYSD, wherein X₂ is A or H (SEQID NO: 5); (iv) CDRL1 comprises the amino acid sequence of SEQ ID NO: 6;(v) CDRL2 comprises the amino acid sequence of SEQ ID NO: 7; and (vi)CDRL3 comprises the amino acid sequence of AX₃GTYYPHT, wherein X₃ is Qor H (SEQ ID NO: 8), wherein at least one of X₁, X₂, and X₃ is H, themethod comprising: culturing a host cell comprising a firstpolynucleotide encoding the heavy chain variable region and a secondpolynucleotide encoding the light chain variable region under suitableconditions such that the heavy chain variable region and light chainvariable region are expressed, and the antibody is produced.
 44. Amethod for inhibiting the activity of ApoC3 in a subject, the methodcomprising administering to the subject an effective amount of anantibody that specifically binds to ApoC3, the antibody comprising aheavy chain variable region comprising complementarity determiningregions CDRH1, CDRH2 and CDRH3, and a light chain variable regioncomprising complementarity determining regions CDRL1, CDRL2 and CDRL3,wherein: (a) CDRH1 comprises the amino acid sequence of SEQ ID NO: 3;(b) CDRH2 comprises the amino acid sequence of SIX₁TDGGGTAYRDSVKG,wherein X₁ is S or H (SEQ ID NO: 4); (c) CDRH3 comprises the amino acidsequence of X₂GYSD, wherein X₂ is A or H (SEQ ID NO: 5); (d) CDRL1comprises the amino acid sequence of SEQ ID NO: 6; (e) CDRL2 comprisesthe amino acid sequence of SEQ ID NO: 7; and (f) CDRL3 comprises theamino acid sequence of AX₃GTYYPHT, wherein X₃ is Q or H (SEQ ID NO: 8),and wherein at least one of X₁, X₂, and X₃ is H. 45.-62. (canceled) 63.The polynucleotide of claim 40, wherein: (a) the CDRH2 comprises anamino acid sequence selected from the group consisting of SEQ ID NOs: 9and 11; (b) the CDRH3 comprises an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 10 and 12; and/or (c) the CDRL3comprises an amino acid sequence selected from the group consisting ofSEQ ID NOs: 13 and
 14. 64. The polynucleotide of claim 40, wherein theCDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 comprise the amino acidsequences set forth in SEQ ID NOs: 3, 11, 10, 6, 7, and 13; 3, 9, 12, 6,7, and 13; 3, 9, 10, 6, 7, and 14; 3, 11, 10, 6, 7, and 14; 3, 9, 12, 6,7, and 14; 3, 11, 12, 6, 7, and 13; or 3, 11, 12, 6, 7, and 13,respectively.
 65. The polynucleotide of claim 40, wherein the heavychain variable region comprises an amino acid sequence that is at least75% identical to the amino acid sequence set forth in SEQ ID NO: 15, 16,17, or 18, and/or the light chain variable region comprises an aminoacid sequence that is at least 75% identical to the amino acid sequenceset forth in SEQ ID NO: 19 or
 20. 66. The polynucleotide of claim 40,wherein the heavy chain variable region and the light chain variableregion, respectively, comprise amino acid sequences that are each atleast 75% identical to the amino acid sequences set forth in SEQ ID NOs:16 and 19, 17 and 19, 18 and 19, 15 and 20, 16 and 20, 17 and 20, or 18and
 20. 67. The polynucleotide of claim 40, wherein the heavy chainvariable region and the light chain variable region, respectively,comprise the amino acid sequences set forth in SEQ ID NOs: 16 and 19, 17and 19, 18 and 19, 15 and 20, 16 and 20, 17 and 20, or 18 and
 20. 68.The polynucleotide of claim 40, wherein the antibody further comprises ahuman or humanized constant region.
 69. The polynucleotide of claim 68,wherein the constant region is a variant of a wild type humanimmunoglobulin heavy chain constant region, and wherein the varianthuman immunoglobulin heavy chain constant region has an increasedaffinity for human neonatal Fc receptor (FcRn) at pH 6 relative to theaffinity of the wild type human immunoglobulin heavy chain constantregion for human FcRn at pH
 6. 70. The polynucleotide of claim 68,wherein the constant region is a heavy chain constant region of a humanIgG, optionally a heavy chain constant region of a human IgG₁, IgG₂, orIgG₄.
 71. The polynucleotide of claim 68, wherein the constant regioncomprises: (a) the amino acids K, F, and Y at EU positions 433, 434, and436, respectively; (b) the amino acids Y, T, and E at EU positions 252,254, and 256, respectively; or (c) the amino acids L and S at EUpositions 428 and 434, respectively, optionally wherein the constantregion comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 22-24, 37-39, and 42-47.